Use of Photopolymerization for Amplification and Detection of a Molecular Recognition Event

ABSTRACT

The invention provides methods to detect molecular recognition events. The invention also provides methods to detect the presence of or identify a target species based on its interaction with one or more probe species. The methods of the invention are based on amplification of the signal due to each molecular recognition event. The amplification is achieved through photopolymerization, with the polymer formed being associated with the molecular recognition event. In one aspect, a fluorescent polymer, a magnetic polymer, a radioactive polymer or an electrically conducting polymer can form the basis of detection and amplification. In another aspect, a polymer gel swollen with a fluorescent solution, a magnetic solution, a radioactive solution or an electrically conducting solution can form the basis of detection and amplification. In another aspect, detectable particles can be included in the polymer formed. In another aspect, sufficient polymer forms to be detectable by visual inspection.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. application Ser. No.11/372,485, filed Mar. 9, 2006. This application also claims the benefitof U.S. provisional applications 60/988,563, filed Nov. 16, 2007 and60/982,992, filed Oct. 26, 2007. U.S. application Ser. No. 11/372,485 isa continuation-in-part of International Application serial numberPCT/US2004/029733, filed Sep. 9, 2004, and claims the benefit of U.S.provisional application Ser. No. 60/662,313, filed Mar. 16, 2005;International Application PCT/US2004/029733 claims the benefit of U.S.provisional application Ser. No. 60/501,755, filed on Sep. 9, 2003. Allof these applications are hereby incorporated by reference to the extentnot inconsistent with the disclosure herein.

ACKNOWLEDGEMENT OF GOVERNMENT SUPPORT

This invention was made at least in part with support from the NationalInstitutes of Health under grant numbers NIH HG003100, R41 AI060057 andSGER 0442047, the US Air Force under grant number AFOSR F49620-02-1-0042and the US Air Force under grant number AFOSR F49620-02-1-0042. TheUnited States Government has certain rights in this invention.

BACKGROUND OF THE INVENTION

This invention is in the field of detection of molecular recognitionevents, in particular use of photopolymerization for amplification anddetection of these events.

A variety of methods exist for detection of molecular recognitionevents. Detection of molecular recognition events such as DNAhybridization, antibody-antigen interactions, and protein-proteininteractions becomes increasingly difficult as the number of recognitionevents to be detected decreases. Of particular interest are molecularrecognition events between a target and a probe.

One approach to the problem is to increase the number of recognitionevents taking place. For example, polymerase chain reaction (PCR)increases the number of copies of DNA or RNA to be detected. Othermolecular biology techniques which increase the number of copies of DNAor RNA to be detected include reverse transcription polymerase chainreaction (RT-PCR), strand displacement amplification, and Eberwinelinear amplification.

Another approach is to amplify the signal due to each molecularrecognition event. For example, DNA detection methods based onoligonucleotide-modified particles have been reported (U.S. Pat. Nos.6,740,491, 6,777,186, 6,773,884, 6,767,702, 6,759,199, 6,750,016,6,730,269, 6,720,411, 6,720,147, 6,709,825, 6,682,895, 6,673,548,6,667,122, 6,645,721, 6,610,491, 6,582,921, 6,506,564, 6,495,324,6,417,340 and 6,361,944 and Park, S.-J. et al, 2002, Science, 295, 5559,1503-1506). U.S. Pat. No. 6,602,669 relates to silver stainingnanoparticles.

DNA detection methods based on branched DNA have also been reported(U.S. Pat. Nos. 5,681,702, 5,597,909, 5,580,731, 5,359,100, 5,124,246,5,545,730, 5,594,117, 5,571,670, 5,594,118, 5,681,697, 5,591,584,5,571,670, 5,624,802, 5,635,352, and 5,591,584. The branched DNA assayis a solution phase assay that involves a number of probeoligonucleotides that bind to multiple sites on the target viral RNA.Detection is possible because each hybridization event is accompanied bythe binding of a fluorophore (Kern, D., Collins, M., Fultz, T., Detmer,J., Hamren, S., Peterkin, J., Sheridan, P., Urdea, M., White, R.,Yeghiazarian, T., Todd, J. (1996) “An Enhanced-sensitivity Branched-DNAAssay for Quantification of Human Immunodeficiency Virus Type 1 RNA inPlasma” Journal of Clinical Microbiology 34:3196-3203). The syntheticeffort required for this assay is relatively large: multiple probes aredesigned for each RNA of interest, and the assay depends on the bindingof these probes to multiple preamplifier and amplifier molecules thatalso must be designed and synthesized.

Dendrimer-based DNA detection methods have also been reported (U.S. Pat.Nos. 5,710,264, 5,175,270, 5,487,973, 5,484,904 and Stears, R. et al.,2000, Physiol. Genomics 3: 93-99). Dendrimers are complexes of partiallydouble-stranded oligonucleotides, which form stable, sphericalstructures with a determined number of free ends. Specificity of thedendrimer detection is accomplished through specific binding of acapture oligonucleotide on a free arm of the dendrimer. Other arms ofthe dendrimer are labeled for detection. This method does not requireenzymes and can produce amplification of 300-400.

Tyramide signal amplification is reported in U.S. Pat. Nos. 6,593,100and 6,372,937.

Rolling circle amplification has been described in the scientificliterature (Baner et al. (1998) Nuc. Acids Res. 26:5073-5078; Barany, F.(1991) Proc. Natl. Acad. Sci. USA 88:189-193; Lizardi et al. (1998),Nat. Genet. 19:225-232; Zhang et al., Gene 211:277 (1998); andDaubendiek et al., Nature Biotech. 15:273 (1997)). Rolling circleamplification is capable of detecting as few as 150 molecules bound to amicroarray (Nallur, G., Luo, C., Fang, L., Cooley, S., Dave, V.,Lambert, J., Kukanskis, K., Kingsmore, S., Lasken, R., Schweitzer, B.(2001) “Signal Amplification by Rolling Circle Amplification on DNAMicroarrays” Nucleic Acids Research 29:E118). The main drawback to RCAis the necessity of DNA polymerase.

Ligase chain reaction is reported in U.S. Pat. Nos. 5,185,243 and5,573,907.

Cycling probe technology is reported in U.S. Pat. Nos. 5,011,769,5,403,711, 5,660,988, and 4,876,187.

Microfabricated disposable DNA sensors based on enzymatic amplificationelectrochemical detection was reported by Xu et al. (Xu et al., 2001,Electoanalysis, 13(10), 882-887).

Surface initiated polymerization from surface confined initiators hasbeen reported. Biesaiski et al. report poly(methyl methacrylate) brushesgrown in situ by free radical polymerization from an azo-initiatormonolayer covalently bound to the surface (Biesalski, M. et al., (1999),J. Chem. Phys., 111(15), 7029). Surface initiated polymerization foramplification of patterned self-assembled monolayers bysurface-initiated ring opening polymerization (Husemann, M. et al.,Agnewandte Chemie Int. Ed. (1999), 38(5) 647-649) and atom transferradical polymerization (Shah, R. R. et al., (2000), Macromolecules, 33,597-605) has been also reported.

WO/2007/095464 to Kuck reports signal amplification of biorecognitionevents using photopolymerization in the presence of air.

DNA microarrays, or biochips, represent promising technology foraccurate and relatively rapid pathogen identification (Wang, D., Coscoy,L., Zylberberg, M., Avila, P. C., Boushey, H. A., Ganem, D., DeRisi, J.L. (2002) “Microarray Based Detection and Genotyping of ViralPathogens,” PNAS, 99(24), 15687-15692). Anthony et al. recentlydemonstrated rapid identification of 10 different bacteria in bloodcultures using a BioChip (Anthony, R. M., Brown, T. J., French, G. L.(2000) “Rapid Diagnosis of Bacteremia by Universal Amplification of 23SRibosomal DNA Followed by Hybridization to an Oligonucleotide Array”Journal of Clinical Microbiology 38:781-788). The microarray assay wasconducted in ˜4 hrs. The approach utilized universal primers for PCRamplification of the variable region of bacterial 23s ribosomal DNA, anda 3×10 array of 30 unique capture sequences. This work demonstrates animportant aspect of BioChip platforms—the capability to screen formultiple pathogens simultaneously. DeRisi and co-workers demonstrated a“virus chip” that contained sequences for hundreds of viruses, includingmany that cause respiratory illness (Wang et al., 2002). This chipproved useful in identifying the corona virus associated with SARS(Risberg, E. (2003) “Gene Chip Helps Identify Cause of Mystery Illness,”USA Today (Jun. 18, 2003)). Evans and co-workers have demonstrated thata DNA microarray could be used for typing and sub-typing human influenzaA and B viruses (Li, J., Chen, S., & Evans, D. H. (2001) “Typing andSubtyping Influenza Virus Using DNA Microarrays and Multiplex ReverseTranscriptase PCR” Journal of Clinical Microbiology 39:696-704). In boththe DeRisi and Evans work PCR technology was used to amplify the geneticmaterial for capture and relatively expensive fluorescent labels (˜$50in labels per chip) were used to generate signals from positive spots.Townsend et al. report experimental evaluation of a FluChip diagnosticmicroarray for influenza virus surveillance (Townsend, M. et al., J.Clinical Microbiology, August 2006, 44(8), 2863-2871). Dawson et al.report DNA microarrays that target the matrix gene segment of influenzaA (MChip) (Dawson, E. et al., October 2006, Anal. Chem., 78(22),7610-7615; Dawson, E. et al, November 2006, Anal. Chem., 79 (1),378-384, 2007).

There remains a need in the art for relatively inexpensive labeling andsignal amplification methods for molecular recognition events which donot require the use of enzymes for amplification. These methods would beuseful in combination with DNA microarrays.

SUMMARY OF THE INVENTION

In an embodiment, the invention provides methods to detect molecularrecognition events, in particular a relatively small number of molecularrecognition events. The methods of the invention are based onamplification of the signal due to each molecular recognition event,rather than amplification of the number of molecular recognition eventstaking place. The present invention can limit or eliminate the need fortechniques which increase the number of recognition events taking place,including PCR and techniques involving culturing of bacteria. Thepresent invention can replace PCR and RT-PCR techniques for microarrayapplications as a means to achieve acceptable signals.

In general, the methods of the invention can be used to generate andamplify a signal due to many types of molecular recognition events thatcan be described by the following equation:

A+B+In→A−B−In  (Eqn. 1)

where A and B are the species of interest that undergo molecularrecognition and In is a photoinitiator. A is the probe species and B isthe target species. For a microarray, the probe A is attached to thesubstrate. The target species, B, and/or the photoinitiator may comprisea linking group which allows selective binding of the photoinitiator tothe target or the A−B complex. As an example, the target species maycomprise biotin and the initiator avidin. In an embodiment, the targetspecies comprises one of biotin and a biotin-binding protein and theprobe species comprises the other of biotin and a biotin-bindingprotein. Biotin-binding proteins include avidin, streptavidin, andNeutravidin (a deglycosylated form of avidin).

When the initiator comprises a linking group, Equation 1 may also bewritten as:

A+B+C−In→A−B−C−In  (Eqn. 2)

where C comprises an entity which allows selective binding of thephotoinitiator to the target or the A−B complex.

In an embodiment, the molecular recognition event occurs between atarget and a probe to form a target-probe complex. The target-probecomplex is labeled with a photoinitiator label which comprises aphotoinitiator. In one embodiment, the photoinitiator is capable ofbeing activated by ultraviolet (UV) light and photopolymerization isinitiated by exposure to a source of UV light. In this embodiment, aco-initiator may not be required. In another embodiment, thephotoinitiator is capable of being activated by visible light andphotopolymerization is initiated by exposure to a source of visiblelight. In an embodiment, the photoinitiator is part of a two-partphotoinitiator system comprising a photoinitiator and a co-initiator. Inan embodiment, the photoinitiator interacts with the co-initiator togenerate free-radicals upon exposure to a source of visible light.

The amplification scheme relies on the large number of propagationevents that occur for each initiation event. Depending on the specificpolymerization system used (light intensity, initiator concentration,monomer formulation, temperature, etc.), each initiator can lead to thepolymerization of as many as 10²-10⁶ monomer units. Thus, each singlemolecular recognition event has the opportunity to be amplified by thepolymerization of up to 10⁶ or 10⁷ monomers, each of which may befluorescent or enable detection of its presence through one of a varietyof means. In other embodiments, the detectable response can be generatedfrom as low as 10⁴, 10⁵, or 10⁶ molecular recognition events.

In an embodiment, a fluorescent polymer, a magnetic polymer, aradioactive polymer or an electrically conducting polymer can form thebasis of detection and amplification. In another embodiment, a polymergel swollen with a fluorescent solution, a magnetic solution, aradioactive solution or an electrically conducting solution can form thebasis of detection and amplification. In another embodiment, a polymercontaining a plurality of detectable particles can form the basis ofdetection and amplification. In an embodiment, the particles may bedetected on the basis of fluorescence, magnetic properties,radioactivity, electrical conductivity, or light absorption/color.

In another embodiment, the quantity of polymer formed is sufficient toallow visual detection of polymer formation. In this embodiment, thepolymer need not be fluorescent, magnetic, radioactive or electricallyconducting. This embodiment can be achieved through a synergisticcombination of reduction of oxygen content in the polymer precursorsolution by purging, utilization of a photoinitiator label with anappropriate ratio of initiator to molecular recognition agent, and theidentification of the appropriate exposure time. Without wishing to bebound by any particular belief, it is believed that a process having allthese attributes can yield much higher degrees of amplification andenable better contrast than is possible with a process having only oneof these attributes.

In an embodiment, the invention provides a method for amplifying amolecular recognition interaction between a target and a probecomprising the steps of:

-   -   a. contacting the target with the probe under conditions        effective to form a target-probe complex;    -   b. removing target not complexed with the probe;    -   c. contacting the target-probe complex with a photoinitiator        label under conditions effective to attach the photoinitiator        label to the target-probe complex;    -   d. removing photoinitiator label not attached to the        target-probe complex;    -   e. contacting the photoinitiator-labeled target-probe complex        with a polymer precursor solution;    -   f. exposing the photoinitiator-labeled target-probe complex and        the polymer precursor solution to light, thereby forming a        polymer; and    -   g. detecting the polymer formed, thereby detecting an amplified        target-probe interaction.

In an embodiment, the invention provides a method for amplifying amolecular recognition interaction between a target and a probecomprising the steps of:

-   -   a. contacting the target with the probe under conditions        effective to form a target-probe complex;    -   b. removing target not complexed with the probe;    -   c. contacting the target-probe complex with a photoinitiator        label under conditions effective to attach the photoinitiator        label to the target-probe complex;    -   d. removing photoinitiator label not attached to the        target-probe complex;    -   e. contacting the photoinitiator-labeled target-probe complex        with a polymer precursor solution;    -   f. exposing the photoinitiator-labeled target-probe complex and        the polymer precursor solution to light, thereby forming a        polymer; and    -   g. detecting the polymer formed, thereby detecting an amplified        target-probe interaction,        wherein the photoinitiator label is a macroinitiator and the        oxygen content of the polymer precursor solution during light        exposure is sufficiently low and the time of light exposure is        sufficiently long that the polymer forms in sufficient        quantities to allow visual detection.

In another embodiment, the invention provides methods for identificationof a target species based on its molecular interaction with an array ofdifferent probe species, each probe species being attached to a solidsubstrate at known locations. In the methods of the invention, if thetarget species undergoes a molecular recognition reaction with a probe,the probe will be labeled with a polymer. Detection of thepolymer-labeled probes allows identification of which probes haveundergone the molecular recognition reaction and thereforeidentification of the target.

In an embodiment, the invention provides a method for identifying atarget comprising the steps of:

-   -   a. providing a probe array comprising a plurality of different        probes, wherein the probes are attached to a solid substrate at        known locations;    -   b. contacting the probe array with the target under conditions        effective to form a target-probe complex;    -   c. removing target not complexed with the probe;    -   d. contacting the target-probe complex with a photoinitiator        label under conditions effective to attach the label to the        target-probe complex;    -   e. removing photoinitiator label not attached to the        target-probe complex;    -   f. contacting the photoinitiator-labeled target-probe complex        with a polymer precursor solution;    -   g. exposing the photoinitiator-labeled target-probe complex and        the polymer precursor to light, thereby forming a polymer; and    -   h. detecting the polymer formed, wherein the polymer location        indicates the probe which forms a target-probe complex with the        target, thereby identifying the target.

In an embodiment, the invention provides a method for identifying atarget comprising the steps of:

-   -   a. providing a probe array comprising a plurality of different        probes, wherein the probes are attached to a solid substrate at        known locations;    -   b. contacting the probe array with the target under conditions        effective to form a target-probe complex;    -   c. removing target not complexed with the probe;    -   d. contacting the target-probe complex with a photoinitiator        label under conditions effective to attach the label to the        target-probe complex;    -   e. removing photoinitiator label not attached to the        target-probe complex;    -   f. contacting the photoinitiator-labeled target-probe complex        with a polymer precursor solution;    -   g. exposing the photoinitiator-labeled target-probe complex and        the polymer precursor solution to light, thereby forming a        polymer; and    -   h. detecting the polymer formed, wherein the polymer location        indicates the probe which forms a target-probe complex with the        target, thereby identifying the target,        wherein the photoinitiator label is a macroinitiator and the        oxygen content of the polymer precursor solution during light        exposure is sufficiently low and the time of light exposure is        sufficiently long that the polymer forms in sufficient        quantities to allow visual detection.

In another embodiment, the invention provides a method for identifying atarget comprising the steps of providing a probe array comprising aplurality of different probes, wherein the probes are attached to asolid substrate at known locations; the method comprising the steps of:

-   -   a. contacting the target with the probe array under conditions        effective to form a target-probe complex;    -   b. removing target not complexed with the probe;    -   c. contacting the target-probe complex with a photoinitiator        label under conditions effective to attach the photoinitiator        label to the target-probe complex, the photoinitiator label        comprising a photoinitiator capable of being activated by        exposure to UV light;    -   d. removing photoinitiator label not attached to the        target-probe complex;    -   e. contacting the photoinitiator-labeled target-probe complex        with a polymer precursor solution;    -   f. exposing the photoinitiator-labeled target-probe complex and        the polymer precursor solution to UV light, thereby forming a        polymer; and    -   g. detecting the polymer formed,        wherein the location of the polymer formed indicates the probe        which forms a target-probe complex with the target, thereby        identifying the target and the oxygen content of the polymer        precursor solution during step f) is limited by contacting the        polymer precursor solution with a purge gas prior to step e),        during step e), during step f), or combinations thereof.

In another embodiment, the invention provides a method for identifying atarget comprising the steps of providing a probe array comprising aplurality of different probes, wherein the probes are attached to asolid substrate at known locations; the method comprising the steps of:

-   -   a. contacting the target with the probe array under conditions        effective to form a target-probe complex;    -   b. removing target not complexed with the probe;    -   c. contacting the target-probe complex with a photoinitiator        label under conditions effective to attach the photoinitiator        label to the target-probe complex, the photoinitiator label        comprising a photoinitiator capable of being activated by        exposure to visible light;    -   d. removing photoinitiator label not attached to the        target-probe complex;    -   e. contacting the photoinitiator-labeled target-probe complex        with a polymer precursor solution comprising a polymer precursor        and a co-initiator;    -   f. exposing the photoinitiator-labeled target-probe complex and        the polymer precursor solution to visible light, thereby forming        a polymer; and    -   g. detecting the polymer formed,        wherein the location of the polymer formed indicates the probe        which forms a target-probe complex with the target, thereby        identifying the target and the oxygen content of the polymer        precursor solution during step f) is limited by contacting the        polymer precursor solution with a purge gas prior to step e),        during step e), during step f), or combinations thereof.

The methods of the invention can be used to identify target speciesusing DNA microarrays, also commonly referred to as DNA chips orBioChips, to provide the probe array. In an embodiment, the DNAmicroarray may be an array which allows identification and strainanalysis for one or more genera of influenza virus.

In one aspect of the invention, observation of polymer formation can beused as a qualitative indicator of the presence or absence of thetarget. The threshold for such a yes/no response is tunable. Forexample, for probes attached to a substrate the threshold can be tunedby adjusting the probe concentration on the substrate surface.

In another aspect of the invention, the amount of target can bedetermined quantitatively. In an embodiment, the amount of target can bedetermined through measurement of a detectable characteristic of thepolymer formed. Suitable detectable characteristics include, but are notlimited to, the amount of polymer formed, the thickness of polymerformed fluorescence, magnetic properties, radioactivity, electricalconductivity and adsorption. In an embodiment, the amount of target canbe determined through use of a reference correlation between the amountof target (or a target test species) and the value of the detectablecharacteristic. For example, the amount of target may be determined fromthe thickness of the polymer formed when the probe molecules areattached to a substrate in “spots” of known size. In this case, thecorrelation referred to can relate polymer film thickness toconcentration of target or to a test species.

In another embodiment, the amount of target can be determined throughmeasurement of a detectable characteristic of one or more additionaldetectable components contained within the polymer formed. Suitabledetectable components include, but are not limited to fluorescent,magnetic, radioactive, electrically conducting, or absorptive/coloredparticles. In an embodiment, the amount of target can be determinedthrough use of a reference correlation between the amount of target (ora target test species) and the value of the detectable characteristic ofthe detectable component. For example, the amount of target may bedetermined from analysis of the amount of fluorescence observed fromfluorescent particles incorporated into the polymer.

In another embodiment, the amount of target can be determined throughanalysis of the polymerization parameters required to obtain a givenvalue for a particular characteristic of the polymer or of detectablecomponents contained within the polymer. In an embodiment, thepolymerization conditions to obtain a selected value of a detectablecharacteristic of the polymer can be compared to a reference correlationor other reference information. For example, the minimum time orradiation dose to obtain sufficiently thick polymer for visualobservation can be used to determine the concentration of target when acalibration of visualization time versus concentration of target or testspecies is available.

In an embodiment, the methods of the invention provide sufficientamplification that molecular recognition can be detected withoutinstrumentation. In another embodiment, the methods of the inventionprovide sufficient amplification that molecular recognition can bedetected using a relatively inexpensive microarray reader or scannerwhich may not have the highest instrument sensitivity or resolution.

BRIEF SUMMARY OF THE FIGURES

FIG. 1 schematically illustrates some of the steps in detection andamplification of hybridization using photopolymerization.

FIG. 2 illustrates an alternate two-step hybridization scheme fordetection and amplification.

FIGS. 3A and 3B illustrate fluorescence detection of macroinitiators ona biotin array. FIG. 3A is an image of the array after polymerization;

FIG. 3B shows negative control spots on the same array.

FIG. 4 illustrates an image of a FluChip developed usingphotopolymerization for signal enhancement.

FIG. 5 shows a profilometry scan across a row of polymer spots that weregrown from an array following recognition of biotin by a streptavidinmacrophotoinitiator.

FIGS. 6 a-6 c illustrate polymerization results for a 3×3 array ofbiotinylated oligomers; each spot has a different oligomerconcentration. FIG. 6 a shows an image of the array after polymerizationwith a macrophotoinitiator. FIG. 6 b shows the maximum number ofbiotinylated oligomers present in each spot.

FIG. 6 c shows the minimum radiation dose delivered to each spot priorto observation of polymer formation.

FIGS. 7 a and 7 b illustrate profilometry scans across spots containing3′biotin labeled capture sequences (a) and unlabeled capture sequences(b). Both rows contained capture sequences at a surface density of 10²capture sequences/μm².

FIGS. 8 a and 8 b show film thicknesses obtained for rows containingdifferent amounts of biotinylated DNA on a dilution chip using a visiblelight initiation system.

FIG. 9 shows polymer film thickness as a function of oligonucleotidetarget density for two different light exposure times.

FIG. 10 a shows fluorescent intensity versus biotinylated capturesurface density (molecules/μm²) for various photoinitiator labelingsolution concentrations.

FIG. 10 b shows polymer film thickness versus oligonucleotide targetdensity (molecules/μm²) for various photoinitiator labeling solutionconcentrations.

FIG. 11 compares the measured absorbance from a solution containing thestreptavidin functionalized initiator (SA-EITC) and from fluorophorescontained in the polystyrene nanospheres in monomer solution.

FIG. 12 shows a FTIR conversion plot of hydrogel precursor (25 wt %PEGDA, 225 mM MDEA, 37.5 mM 1-vinyl-2-pyrrolidinone, pH 9.0) atdifferent concentrations of fluorescent nanoparticles and photoinitiatorusing a 495-650 nm light source at 10 mW/cm² A) 2 μM eosin (85 μg/mLSA-EITC), 0 nM fluorescent nanoparticles B) 2 μM eosin, 90 nMfluorescent nanoparticles C) 2 μM eosin, 480 nM fluorescentnanoparticles D) 0 μM SA-EITC, 480 nM fluorescent nanoparticles E) 0 μMSA-EITC, 0 nM fluorescent nanoparticles.

FIG. 13 a shows polymer film thicknesses versus fluorescent signalgenerated from encapsulated fluorescent nanospheres after inclusion inmonomer at 90 or 500 nM concentrations.

FIG. 13 b shows measured fluorophore density verses biotin-SA-EITCbinding events after polymerization-based amplification.

DETAILED DESCRIPTION OF THE INVENTION

FIG. 1 is a conceptual diagram of how photopolymerization of afluorescent monomer M is used to generate and amplify a signal from asingle captured genetic target on a DNA microarray. FIG. 1 showsaddition of biotin (2) to the target oligonucleotide (10) to form abiotinylated target nucleotide (12). The biotinylated target nucleotide(12) is hybridized to a complementary probe (20), forming a target-probecomplex (30) on the surface of the microarray (40). The microarray shownin FIG. 1 contains a biotin-labeled probe (22) which acts as a positivecontrol. After hybridization, the microarray is exposed to aphotoinitiator label (50), initiator-functionalized avidin, whichinteracts with the biotinylated target oligonucleotide (12) and controlprobe (22) to form an initiator-labeled target-probe complex (35) and aninitiator-labeled positive control probe (25). The microarray surface isthen exposed to a fluorescent monomer (M) under the appropriateinitiation conditions. In the presence of light and a fluorescentmonomer (represented by M in FIG. 1), a polymerization reaction occursfrom sites on the surface where targets have been captured by the probeDNA, forming a polymer-labeled target-probe complex (37) and apolymer-labeled positive control probe (27). In FIG. 1, the polymerlabel is denoted by (60). Ideally, since initiators are not bound tosites where hybridization has not occurred, polymerization does notoccur from those sites. For brevity, FIG. 1 omits several steps whichare typically used in the process, including removal of uncomplexedtarget material prior to exposure of the microarray to initiatorfunctionalized avidin, removal of initiator functionalized avidin notattached to the target-probe complex, and removal of unpolymerizedmonomer prior to detection.

Although FIG. 1 illustrates hybridization of complementary DNA to a DNAmicroarray as a specific example, the detection and amplification schemegeneralizes to many other types of molecular recognition events. Agentscapable of participating in molecular recognition events include, butare not limited to, agonists and antagonists for cell membranereceptors, toxins and venoms, viral epitopes, hormones (e.g., opiates,steroids, etc.), hormone receptors, peptides, enzymes, enzymesubstrates, substrate analogs, transition state analogs, cofactors,drugs, proteins, and antibodies, cell membrane receptors, monoclonalantibodies and antisera reactive with specific antigenic determinants(such as on viruses, cells or other materials), drugs, polynucleotides,nucleic acids, peptides, cofactors, lectins, sugars, polysaccharides,cells, cellular membranes, and organelles. In different embodiments, thedetection and amplification scheme can be used to detect and amplify themolecular recognition interaction between nucleic acids, an antibody andan antigen, and a first and a second protein. Microarrays can be used todetect hybridization as well as protein-protein interactions, proteindrug binding, and enzymatic catalysis (Schena, M., “Microarray Analysis,(2003) John Wiley & Sons, New Jersey, p. 153). As used herein, molecularrecognition interactions are those in which the probe recognizes andselectively binds a target, resulting in a target-probe complex.Molecular recognition interactions also involve the formation ofnoncovalent bonds between the two species. The binding occurs betweenspecific regions of atoms (molecular domains) on the probe species whichhave the characteristic of binding or attaching specifically to uniquemolecular domains on specific target species. Molecular recognitioninteractions can also involve responsiveness of one species to anotherbased on the reciprocal fit of a portion of their molecular shapes.

The target and probe are two species of interest which undergo molecularrecognition. The target may also be referred to as a ligand. The probemay also be referred to as a receptor. In an embodiment, at least somecharacteristics of the probe are known. In an embodiment, the probe isan oligonucleotide whose sequence is known or partially known. In otherembodiment, the sequence of the probe may not be known, but it is knownto be complementary to a possible target species. Typically, the probewill be selected so that it is capable of selected recognition with theknown or suspected identity of the target. In some cases a single probecan be used to detect the presence of a target. In other cases more thanone probe will be necessary to detect the presence of or identify atarget.

In order for molecular interaction between the target and the probe toidentify the target, the molecular interaction between the target andthe probe must be sufficiently specific. For hybridization, theselectivity is a measure of the specificity of the molecular recognitionevent. “Selectivity” or “hybridization selectivity” is the ratio of theamount of hybridization (i.e., number of second nucleic acidshybridized) of fully complementary hybrids to partially complementaryhybrids, based on the relative thermodynamic stability of the twocomplexes. For the purpose of this definition it is presumed that thisratio is reflected as an ensemble average of individual molecularbinding events. Selectivity is typically expressed as the ratio of theamount of hybridization of fully complementary hybrids to hybrids havingone base pair mismatches in sequence. Selectivity is a function of manyvariables, including, but not limited to: temperature, ionic strength,pH, immobilization density, nucleic acid length, the chemical nature ofthe substrate surface and the presence of polyelectrolytes and/or otheroligomers immobilized on the substrate or otherwise associated with theimmobilised film.

For hybridization, the homology of the target and probe moleculesinfluences whether hybridization occurs. Cross-hybridization can occurif the sequence identity between the target and the probe is greaterthan or equal to about 70% (Schena, M., “Microarray Analysis, (2003)John Wiley & Sons, New Jersey, p. 151).

In an embodiment, either the target or the probe is a nucleic acid. Inan embodiment, both the target and the probe are a single strandednucleic acid. In an embodiment, the probe is an oligonucleotide, arelatively short chain of single-stranded DNA or RNA. “Nucleic acid”includes DNA and RNA, whether single or double stranded. The term isalso intended to include a strand that is a mixture of nucleic acids andnucleic acid analogs and/or nucleotide analogs, or that is made entirelyof nucleic acid analogs and/or nucleotide analogs and that may beconjugated to a linker molecule. “Nucleic acid analogue” refers tomodified nucleic acids or species unrelated to nucleic acids that arecapable of providing selective binding to nucleic acids or other nucleicacid analogues. As used herein, the term “nucleotide analogues” includesnucleic acids where the internucleotide phosphodiester bond of DNA orRNA is modified to enhance bio-stability of the oligomer and “tune” theselectivity/specificity for target molecules (Uhlmann, et al., (1990),Angew. Chem. Int. Ed. Eng., 90: 543; Goodchild, (1990), J. BioconjugateChem., I: 165; Englisch et al., (1991), Angew, Chem. Int. Ed. Eng., 30:613). Such modifications may include and are not limited tophosphorothioates, phosphorodithioates, phosphotriesters,phosphoramidates or methylphosphonates. The 2′-O-methyl, allyl and2′-deoxy-2′-fluoro RNA analogs, when incorporated into an oligomer showincreased biostability and stabilization of the RNA/DNA duplex (Lesniket al., (1993), Biochemistry, 32: 7832). As used herein, the term“nucleic acid analogues” also include alpha anomers (α-DNA), L-DNA(mirror image DNA), 2′-5′ linked RNA, branched DNA/RNA or chimeras ofnatural DNA or RNA and the above-modified nucleic acids. For thepurposes of the present invention, any nucleic acid containing a“nucleotide analogue” shall be considered as a nucleic acid analogue.Backbone replaced nucleic acid analogues can also be adapted to for useas immobilized selective moieties of the present invention. For purposesof the present invention, the peptide nucleic acids (PNAs) (Nielsen etal., (1993), Anti-Cancer Drug Design, 8: 53; Engels et al., (1992),Angew, Chem. Int. Ed. Eng., 31: 1008) and carbamate-bridgedmorpholino-type oligonucleotide analogs (Burger, D. R., (1993), J.Clinical Immunoassay, 16: 224; Uhlmann, et al., (1993), Methods inMolecular Biology, 20, “Protocols for Oligonucleotides and Analogs,” ed.Sudhir Agarwal, Humana Press, NJ, U.S.A., pp. 335-389) are also embracedby the term “nucleic acid analogues”. Both exhibit sequence-specificbinding to DNA with the resulting duplexes being more thermally stablethan the natural DNA/DNA duplex. Other backbone-replaced nucleic acidsare well known to those skilled in the art and can also be used in thepresent invention (See e.g., Uhlmann et al., (1993), Methods inMolecular Biology, 20, “Protocols for Oligonucleotides and Analogs,” ed.Sudhir Agrawal, Humana Press, NJ, U.S.A., pp. 335).

More generally, the probe and/or target can be an oligomer. “Oligomer”refers to a polymer that consists of two or more monomers that are notnecessarily identical. Oligomers include, without limitation, nucleicacids (which include nucleic acid analogs as defined above),oligoelectrolytes, hydrocarbon based compounds, dendrimers, nucleic acidanalogues, polypeptides, oligopeptides, polyethers, oligoethers any orall of which may be immobilized to a substrate. Oligomers can beimmobilized to a substrate surface directly or via a linker molecule.

In an embodiment, the probe is DNA. The DNA may be genomic DNA or clonedDNA. The DNA may be complementary DNA (cDNA), in which case the targetmay be messenger RNA (mRNA). The DNA may also be an Expressed SequenceTag (EST) or a Bacterial Artificial Chromosome (BAC). For use inhybridization microarrays, double-stranded probes are denatured prior tohybridization, effectively resulting in single-stranded probes.

DNA microarrays are known to the art and commercially available. Thegeneral structure of a DNA microarray is a well defined array of spotson an optically flat surface, each of which contains a layer ofrelatively short strands of DNA. As referred to herein, microarrays havea spot size less than about 1.0 mm. In most hybridization experiments,15-25 nucleotide sequences are the minimum oligonucleotide probe length(Schena, M., “Microarray Analysis, (2003) John Wiley & Sons, New Jersey,p. 8). The substrate is generally flat glass primed with an organosilanethat contains an aldehyde functional group. The aldehyde groupsfacilitate covalent bond formation to biomolecules with free primaryamines via Schiff base interactions. After reaction the chip is cured toform a very stable array ready for hybridization.

Protein microarrays are also known to the art and some are commerciallyavailable. The general structure of protein microarrays can be similarto that of DNA microarrays, except that array spots can containantibodies (in particular monoclonal antibodies), antigens, recombinantproteins, or peptides. For accurate measurement of binding events,surface-bound proteins must be correctly folded and fully functional(Constans, A., 2004, The Scientist, 18(15) 42). To reduce proteinunfolding, the proteins can be protected by use of stabilizing buffersand/or relatively high protein concentrations (Schena, M., “MicroarrayAnalysis, (2003) John Wiley & Sons, New Jersey, p. 154). To avoid theprotein folding problem, the functional domains of interest can bearrayed rather than the whole protein, forming domain-based arrays(Constans, 2004, ibid).

In an embodiment, the target is genetic material from influenza A, B, orC. Influenza is an orthomyxovirus with three genera, types A, B, and C.The types are distinguished by the nucleoprotein antigenicity (Dimmock,N. J., Easton, A. J., Leppard, K. N. (2001) “Introduction to ModernVirology” 5^(th) edition, Blackwell Science Ltd., London). Influenza Aand B each contain 8 segments of negative sense ssRNA. Type A virusescan also be divided into antigenic sub-types on the basis of two viralsurface glycoproteins, hemagglutinin (HA) and neuraminidase (NA). Thereare currently 15 identified HA sub-types (designated H1 through H15) and9 NA sub-types (N1 through N9) all of which can be found in wild aquaticbirds (Lamb, R. A. & Krug, R. M., (1996) “Orthomyxoviridae: The Virusesand their Replication, in Fields Virology”, B. N. Fields, D. M. Knipe,and P. M. Howley, Editors. Lippincott-Raven: Hagerstown). Of the 135possible combinations of HA and NA, only four (H1N1, H1N2, H2N2, andH3N2) have widely circulated in the human population since the virus wasfirst isolated in 1933. The two most common sub-types of influenza Acurrently circulating in the human population are H3N2 and H1N1. LI etal. describe a DNA microarray whose probes were multiple fragments ofthe hemagglutinin, neuraminidase, and matrix protein genes. (Li, J. etal., (2001), J. Clinical Microbio., 39(2), 696-704).

For probes bound to a substrate using aldehyde attachment chemistry, thesubstrate may be treated with an agent to reduce the remaining aldehydesprior to contacting the probe with the target. One suitable reducingagent is sodium borohydride NaBH₄. Such a treatment can decrease theamount of reaction between the monomer and the aldehyde coating on theglass, thus decreasing the amount of background signal during thedetection step.

Prior to contacting the target with the probe, the target may bebiotinylated to allow later attachment of at least one initiator viabiotin-avidin interaction. In an embodiment, photobiotinylation reagents(Pierce, Quanta Biodesign) can be used to biotin-label the target. Forexample, a single-stranded target may be labeled with biotin insolution, allowed to hybridize with a single-stranded probe to form atarget-probe complex, and post-hybridization exposed to UV to crosslinkthe biotinylated target to an aminosilated layer.

The target may also be biotinylated after formation of the target-probecomplex. As an example, biotin may be attached to a target-probe complexpost-hybridization via primer extension with biotin labeleddeoxyribonucleotide triphosphates (dNTPs). In an embodiment, a Klenowfragment uses dsDNA as a primer for 5′->3′ polymerase activity(Pastinen, T. et al, 2000, 10: 1031-1042; Erdogan, F. et al., 2001,29(7), e36).

In an embodiment, the target may be contacted with the photoinitiatorlabel prior to contacting the target with the probe, so long as use of aphotoinitiator-labeled target does not substantially limit itsparticipation in the desired molecular recognition event. In anembodiment, the invention provides a method for amplifying a molecularrecognition interaction between a target and a probe comprising thesteps of contacting a photoinitiator-labeled target with a probe underconditions effective to form a photoinitiator-labeled target-probecomplex, removing target not complexed with the probe, contacting thephotoinitiator-labeled target-probe complex with a polymer precursor,exposing the photoinitiator-labeled target-probe complex and the polymerprecursor to light, thereby forming a polymer, and detecting the polymerformed, thereby detecting an amplified target-probe interaction.

The probe is contacted with a solution comprising the target underconditions effective to form a target-probe complex. The conditionseffective to form a target-probe complex depend on the target and probespecies. For ssDNA or RNA targets binding to ssDNA probes, suitablehybridization conditions have been described in the scientificliterature. In an embodiment, it is sufficient to contact a solutioncomprising the target with the probe for about 2 hours at about 42° C.In an embodiment, this solution also comprises an agent, such as acrowding agent, to limit nonspecific interactions. With reference tonucleic acid interactions, a crowding agent is an agent that interruptsnonspecific adsorption between nucleic acids that are not complementary.Formamide is one such agent to limit nonspecific interactions (Stahl, D.A., and R. Amann. 1991. Development and application of nucleic acidprobes, p. 205-248. In E. Stackebrandt and M. Goodfellow (ed.), Nucleicacid techniques in bacterial systematics. John Wiley & Sons Ltd.,Chichester, United Kingdom). Nonspecific interactions can also belimited by applying a blocking agent to the microarray prior tocontacting the target with the probe. Suitable blocking agents are knownto the art and include, but are not limited to bovine serum albumin(BSA), nonfat milk, and sodium borohydride. Detergents such as sodiumlauroyl sarcosine or sodium dodecyl sulfate can also be added toaldehyde surface hybridization reactions to reduce background (Schena,M., “Microarray Analysis, (2003) John Wiley & Sons, New Jersey, p. 117).The target solution may also be contacted with the probe at highertemperatures in order to limit nonspecific interactions.

After the target is contacted with the probe, targets which have notformed target-probe complexes are removed. The unbound targets can beremoved through rinsing. Water or an aqueous solution may be used forrinsing away unbound targets.

In an embodiment, the substrate surface is treated to minimizenonspecific adsorption of the photoinitiator label. If the initiator isto be attached through biotin-avidin interaction, a blocking agent canbe applied to the microarray to limit nonspecific interaction of avidin.Suitable blocking agents are known to the art and include, but are notlimited to, bovine serum albumin (BSA), nonfat milk and sodiumborohydride. PEG-based blocking agents which react with aminefunctionalities are also known to the art. The blocking agent may beapplied to the substrate surface prior to contact of the photoinitiatorlabel solution with the substrate, may be supplied in the photoinitiatorlabel solution, or both. Denhardt's solution is a commercially availablesolution (Sigma-Aldrich) which contains BSA and can be included in thephotoinitiator label solution. In an embodiment, the array is incubatedwith the blocking agent for approximately 20 minutes at about roomtemperature.

In an embodiment, the target-probe complex is contacted with aphotoinitiator label under conditions effective to attach thephotoinitiator label to the target probe complex. In an embodiment, thetarget-probe complex is contacted with the photoinitiator label bycontacting the target-probe complex with a photoinitiator label solutioncomprising the photoinitiator label. In an embodiment, the solvent isaqueous and the photoinitiator label is water soluble. In an embodiment,the concentration of the photoinitiator label in the solution may beselected to limit nonspecific adsorption of the photoinitiator label tothe surface of a substrate.

In an embodiment, the photoinitiator label comprises a biotin-bindingprotein such as avidin or streptavidin and at least one photoinitiator.In an embodiment, a plurality of photoinitiators is attached to thebiotin binding protein to form a polymeric photoinitiator label. Inanother embodiment, a polymeric photoinitiator label is formed byattaching a plurality of photoinitiators and biotin binding protein to apolymer. In an embodiment, the photoinitiators and biotin bindingprotein are attached to the polymer backbone, for example by attachmentto subunits in the backbone. The polymer to which the photoinitiatorsand biotin binding protein are attached may be chemically the same ordifferent from the polymer formed during exposure of the polymerprecursor solution to light. If the target has been biotin-labeled,interaction between the biotin-binding protein and the biotin can attachthe photoinitiator label to the target, and thus to the target-probecomplex. In another embodiment, both the target and the photoinitiatorcan be labeled with biotin and then multivalent properties of avidin(which can bind four biotins) can be used to bind together the targetand the photoinitiator. Information on avidin-biotin interaction isprovided in Wilcheck, M., (a) Bayer, E. A. Eds. (1990) “Avidin-biotintechnology” Methods in Enzymology 184. In an embodiment, thebiotin-labeled target-probe complex is contacted with a solutioncomprising the photoinitiator label for about 20 minutes at roomtemperature.

Another embodiment suitable for hybridization molecular recognitionevents is schematically illustrated in FIG. 2. In this embodiment, thephotoinitiator label (50) comprises a single strand of DNA attached toat least one photoinitiator (In). The photoinitiator can be attached tothe ssDNA through biotinylation of the DNA followed by interaction withavidin or streptavidin with at least one photoinitiator attached. Inanother embodiment, photoinitiators are coupled directly to the end ofthe oligonucleotide label sequences. Oligonucleotides with 5′ aminemodifications can be purchased and the reaction conditions in Scheme 2(EDC coupling) used to form a peptide bond between this amine and thecarboxylic acid group of the initiator. The product can be purified byHPLC. During the photopolymerization reaction, the target anchors theinitiator, through the label sequence, to the microarray spot. If targetviral RNA will not tolerate the presence of the fluorescent monomer andUV light, it is possible to connect the label sequence to the probeoligonucleotide via a treatment with ligase prior to exposure to thephotopolymerization reaction conditions. Another method that avoids theuse of an enzyme is to place pendant photocrosslinkable groups on theprobe oligonucleotide and the label sequence. If, however, thepolymerization reaction is fast when compared with the timescale ofdiffusion, these steps will not be necessary even if the target geneticmaterial detaches from the capture strand.

A number of photoinitiators are known to the art. Photoinitiators thatare useful in the invention include those that can be activated withlight and initiate polymerization of the polymer precursor. In anembodiment, the photoinitiator is water soluble. Commercially availablephotoinitiators, for example Irgacure 2959 (Ciba), can be modified toimprove their water solubility. In an embodiment, the photoinitiator isa radical photoinitiator. In another embodiment, the photoinitiator is acationic photoinitiator. In another embodiment, the photoinitiatorcomprises a carboxylic acid functional group. The photoinitiator isselected to be compatible with the wavelengths of light supplied.

Photoinitiators include azobisisobutyronitrile, peroxides, phenones,ethers, quinones, acids, formates. Cationic initiators includearyldiazonium, diaryliodonium, and triarylsulfonium salts. In anembodiment, the photoinitiator is selected from the group consisting ofRose Bengal (Aldrich), Darocur or Irgacure 2959(2-hydroxy-1-(4-(hydroxyethoxy)phenyl)-2-methyl-1-propanone, D2959,Ciba-Geigy), Irgacure 651 (2,2-dimethoxy-2-phenylacetophenone, 1651,DMPA, Ciba-Geigy), Irgacure 184 (1-hydroxycyclohexyl phenyl ketone,1184, Ciba-Geigy), Irgacure 907(2-methyl-1-(4-(methylthio)phenyl)-2-(4-morpholinyl)-1-propanone, 1907,Ciba-Geigy), Camphorquinone (CQ, Aldrich), isopropyl thioxanthone(quantacure ITX, Great Lakes Fine Chemicals LTD., Cheshire, England). CQis typically used in conjunction with an amine such as ethyl4-N,N-dimethylaminobenzoate (4EDMAB, Aldrich) or triethanolamine (TEA,Aldrich) to initiate polymerization.

A number of photoinitiators are known to the art which can be activatedby visible light to produce free radicals. In an embodiment, thephotoinitiator is part of a two-part photoinitiator system, comprising aphotoinitiator and a co-initiator. In an embodiment, the photoinitiatorinteracts with the co-initiator to generate free-radicals upon exposureto a source of visible light. In an embodiment, the photoinitiator is aphotoreducible dye. Suitable co-initiators for visible lightphotoinitiators are known to those skilled in the art. Use of visiblelight sources for photoinitiation has the attractive characteristic ofrequiring only a low power, inexpensive and mild excitation source.Further, use of visible light has the added advantage of eliminatingunwanted bulk polymerization which can result from use of UV light. Theuse of visible light, rather than UV light, for photoinitiation can alsoexpand the range of suitable monomer formulations. In an embodiment, themonomer formulation contains high concentrations of bi-functionalmonomers that form thick, highly crosslinked polymer that remains stableon the surface with rinsing. Formation of a surface-stable hydrogelfacilitates characterization of the amplification process with filmthickness and spectroscopic measurements. Finally, visible light canenable more efficient amplification due to its higher penetrationcapability in UV absorbent monomer formulations containing fluorescentmonomers or on UV absorbent surfaces characteristic of glass biochipscontaining surface-bound biomolecules.

In an embodiment, the photoinitiator molecule is an eosin, a brominederivative of fluorescein, or a derivative. In an embodiment, thephotoinitiator molecule is 2′,4′,5′,7′-tetrabromofluorescein or aderivative. In an embodiment, the photoinitiator molecule is Rose Bengalor a derivative. In different embodiments, the photoinitiator isactivated by wavelengths of light between 400 and 700 nm, between 450and 600 nm, between 400 and 500 nm, or between 500 and 600 nm. Suitableco-initiators for fluorescein derivatives include, but are not limitedto, amines such as methyl diethanol amine and tetraethanol amine. In anembodiment where the photoinitiator label comprises eosin attached tostreptavidin, the concentration of the photoinitiator label is 1 μg/mLor less.

Photoinitiator molecules can be attached to avidin or streptavidin bymodification of avidin or streptavidin lysine residues. Forphotoinitiators having a carboxylic acid functional group, thecarboxylic functional group of the photoinitiator can be coupled to theamine of the lysine residue in the presence of a coupling agent. Theresult is the formation of a peptide bond between the initiator and theprotein. Suitable coupling agents are known to those skilled in the artand include, but are not limited to, EDC.

In another embodiment, a polymeric photoinitiator label is formed. Sucha polymeric photoinitiator label can be formed from a polymer which canbe coupled with both the photoinitiator and a molecular recognitiongroup such as avidin or streptavidin. In an embodiment, thephotoinitiator can be attached to the polymer by an ester linkage or byany other kind of linkage known to the art. In an embodiment, the avidinor streptavidin can be attached to the polymer by an amide linkage. Inan embodiment, the polymer comprises carboxylic acid groups and amidegroups. In an embodiment, the polymer comprises a poly(acrylicacid-co-acrylamide) backbone.

In an embodiment, a polymeric photoinitiator label can be formed from apolymer which comprises one part of a two-part photoinitiator system.The polymer is coupled to a molecular recognition group such as avidinor streptavidin. When the combination of the polymer and the second partof the initiator system is exposed to the appropriate wavelength oflight, the initiator system is capable of capable of initiatingpolymerization of a polymer precursor. In an embodiment, one part of thetwo-part photoinitiator system is a tertiary amine which is part of thepolymeric photoinitiator label. The other part of the photoinitiatorsystem can be camphorquinone. (CQ) This two-part system can be activatedby light of approximately 469 nm. The tertiary amine can be incorporatedinto the polymer label by co-polymerizing acrylic acid with a monomercomprising the tertiary amine and an acrylate group.

In an embodiment, the polymeric photoinitiator comprises sufficientphotoinitiators so that it may be regarded as a macroinitiator (havingmany initiators present on a single molecule). The number of initiatorgroups per molecule or chain may vary from one chain to another. In anembodiment, the use of a macroinitiator can increase the averageinitiator concentration by a factor of between about 10 to about 100. Inanother embodiment, the average number of initiators per polymer chainis between about 100 and about 200. In another embodiment, the averagenumber of initiators per polymer chain is between about 120 and about160. The number of molecular recognition groups may also vary from chainto chain. In an embodiment, the average number of molecular recognitiongroups is between one and three. Without wishing to be bound by anyparticular belief, it is believed that the incorporation of too manyinitiator groups can lead to nonspecific interaction between themacroinitiator and the array. The molecular weight of the backbonepolymer is selected to be large enough to allow attachment of theappropriate number of initiator and molecular recognition groups. For apoly(acrylic acid-co-acrylamide) backbone, the molecular weight of thebackbone is preferably greater than about 50,000.

In an embodiment, the polymer backbone of the macroinitiator comprisessufficient hydrophilic monomeric units that the macroinitiator is watersoluble. In an embodiment, the hydrophilic monomeric units are selectedfrom the group consisting of ethylene glycol, acrylate, acrylatederivatives such as acrylamide and hydroxyethylacrylate, and vinylmonomers such as 1-vinyl-2-pyrrolidinone. Without wishing to be bound byany particular belief, hydrophilic macroinitiator backbones are believedto limit nonspecific adsorption of the macroinitiator from aqueoussolutions.

In another embodiment, the photoinitiator label comprises less than 10photoinitiator groups. In an embodiment, the photoinitiator labelincludes from 2 to 9 initiator groups. In another embodiment, thephotoinitiator label comprises from 2 to 3 initiator groups. In anotherembodiment, the average number of initiator groups is from 2 to 3. In anembodiment, the amount of polymer formed using such a photoinitiatorlabel can be used as a quantitative measure of the number of molecularrecognition events

In an embodiment, the photoinitiator label is selected so that thepolymer produced through photopolymerization is bound to the target withsufficient strength that is not easily removed with rinsing (if thetarget is bound to a surface, the polymer can in turn be bound to thesurface). In an embodiment, the photoinitiator molecule is selected sothat the polymer formed is attached to the target through terminationbetween surface stabilized radicals from the photoinitiator and bulkradicals present on the polymer chains. For example, it has beensuggested that eosin radicals are responsible for strong attachment ofpolyethylene glycol (PEG) diacrylate gels onto substrate surfaces(Kizilel, S.; Perez-Luna, V. H.; Teymuor F. Macromolecular Theory andSimulations 2006, 15, 686-700). In one embodiment, the photoinitiatormolecule is fluorescein or a fluorescein derivative such as eosin.

After contact of the photoinitiator label with the target-probe complex,unattached photoinitiator is removed. In an embodiment, photoinitiatorlabel not attached to the target-probe complex is sufficiently removedto reduce any signal resulting from non-specific adsorption toacceptable levels. The excess photoinitiator label may be removed byremoval of the photoinitiator label solution. When the probe is attachedto a solid substrate, the substrate may also be rinsed to remove theexcess. Unattached photoinitiator may be removed by rinsing with wateror an aqueous solution. The rinse may be a room temperature aqueoussolution, such as a TNT solution (1M NaCl, 0.1M Tris, 0.1 wt % Tween20). The rinse may also be at higher temperature, such through exposureto boiling water.

In an embodiment, the photoinitiator-labeled target-probe complex iscontacted with a solution comprising a polymer precursor. As used hereina “polymer precursor” means a molecule or portion thereof which can bepolymerized to form a polymer or copolymer. Polymer precursors includeany substance that contains an unsaturated moiety or other functionalitythat can be used in chain polymerization, or other moiety that may bepolymerized in other ways. Such precursors include monomers andoligomers. In an embodiment, the solution further comprises a solventfor the polymer precursor. In an embodiment, the solvent is aqueous.

The polymer precursor solution may also comprise other components,including molecules which serve to accelerate the polymerizationreaction. In an embodiment, an amine co-initiator is present in aconcentration from 22.5 mM to 2250 mM. In an embodiment, the amineco-initiator is methyl diethanol amine. In an embodiment, an accelerantis present in a concentration from greater than zero to 250 nM. In anembodiment, the accelerator is 1-vinyl-2-pyrrolidinone. In anembodiment, the concentration of vinyl pyrrolidinone and MDEA are 3040mM and 200-250 mM, respectively. In an embodiment, the accelerator is1-vinyl-2-pyrrolidinone. In an embodiment, the initial composition ofpolymer precursor solution does not include photoinitiator.

In an embodiment, the concentration of the monomer components isselected to avoid excessive polymer film thickness, thereby facilitatingquantitative determination of the number of molecular recognition eventsfrom the polymer film thickness. In an embodiment, the monomer solutionis formulated to so that it does not unduly enhance propagation ratesand or minimize termination rates, in contrast to formulations moresuitable for encapsulation applications.

In an embodiment, the backbone of the monomer comprises sufficienthydrophilic monomeric units that the polymer precursor is water soluble.In an embodiment, the hydrophilic monomeric units are selected from thegroup consisting of ethylene glycol, acrylate, acrylate derivatives suchas acrylamide and hydroxyethylacrylate, and vinyl monomers such as1-vinyl-2-pyrrolidinone. In different embodiments, the molecular weightof the polymer is between 200 and 5000 or between 300 and 1000.

In an embodiment, the polymer precursor solution comprises adifunctional polymer precursor. In one embodiment, the amount ofdifunctional polymer is less than 5 wt % of the total weight of thepolymer precursors. In another embodiment the amount of difunctionalpolymer in the solution is from 5 up to 50 wt % (wt % as compared to thesolution as a whole). In different embodiments, the amount ofdifunctional polymer precursor as compared to the total weight ofpolymer precursors in solution is at least 25 wt %, 50 wt %, 75 wt %, or90% wt %. The inclusion of substantial amounts of difunctional monomeris believed to aid in the formation of greater amounts of polymer for agiven polymerization time. For example, the presence of difunctionalacrylate can yield pendant double bonds in propagating polymer chainsthat may crosslink with other propagating chains, thus suppressing chaintermination rates and causing large amounts of high molecular weightpolymer to be generated at the molecular recognition site.

In an embodiment, the solution comprises a difunctional polymerprecursor with acrylate groups at each end. In an embodiment, thedifunctional polymer precursor has a poly(ethylene glycol) (PEG)backbone and acrylate end groups. In an embodiment, the molecular weightof this difunctional PEG monomer is between 300 and 1000. In anembodiment, the weight percent of the difunctional PEG monomer inaqueous solution is from 5% to 50%.

In another embodiment, the solution comprises a mixture of adifunctional monomer with a vinyl group at each end and a monomer with asingle vinyl group. In an embodiment, the polymer precursor solutioncomprises acrylamide and a bis-acrylamide crosslinker such asN,N-methylene-bis-acrylamide. As is known to the art, polymerization ofthese components forms polyacrylamide gel; the structure of the gel(average pore size) is dependent upon the total amount of acrylamidepresent and the relative amount of cross-linker. In an embodiment, thetotal amount of acrylamide and bisacrylamide is 40 wt % in aqueoussolution and 5 mole % of the acrylamide is N,N-methylene-bis-acrylamide.These acrylamide solutions can be used produce thicker polymer coatingsthan some of the PEG solutions. This formulation is compatible withnitrocellulose-coated glass slides which are desirable for antibodyarray testing.

In an embodiment, the pH of the polymer precursor solution is greaterthan 7 and less than or equal to 9. In an embodiment, the pH of thepolymer precursor solution is between 8 and 9. Since the pH of thesolution can affect free radical formation, it is desirable to controlthe pH of the solution during the photopolymerization step.

In another embodiment, the polymer precursor is capable of forming apolymer gel. In an embodiment, the gel is covalently crosslinked and across-linking agent is added to the polymer precursor containingsolution. In another embodiment, the gel is noncovalently crosslinked.In an embodiment, the polymer gel formed is not substantiallyfluorescent, magnetic, radioactive, or electrically conducting. Instead,detection can occur through absorption of a fluorescent, magnetic,radioactive, or electrically conducting solution by the gel. Detectioncan also occur through visual inspection of the quantity of gel formedis sufficiently large.

In an embodiment, the polymer gel is a hydrogel. The term “hydrogel”refers to a class of polymeric materials which are extensively swollenin an aqueous medium, but which do not dissolve in water. In generalterms, hydrogels are prepared by polymerization of a hydrophilic monomerunder conditions where the polymer becomes cross-linked in a threedimensional matrix sufficient to gel the solution. The hydrogel may benatural or synthetic. A wide variety of hydrogel-forming compositionsare known to the art. In an embodiment, the monomer used to form thehydrogel is selected from the group consisting of acrylates,methacrylates, acrylamides, methacrylamides, cyclic lactams and monomerswith ionic functionality. Monomers with ionic functionality includemethacrylate, methacrylamide, and styrene based monomers with acidic orbasic functionality. In an embodiment, the monomer used to form thehydrogel is an acrylate or methacrylate. In another embodiment, thehydrogel-forming monomer is selected from the group consisting ofpolysaccharides and proteins. Polysaccharides capable of forminghydrogels include alginate, chitin, chitosan, cellulose, oligopeptides,and hyalauric acids. Proteins capable of forming hydrogels includealbumin and gelatin. Suitable acrylate mixtures for hydrogel formationinclude, but are not limited to, mixtures of hydroxyethyl acrylate (HEA)and elthylene glycol dimaethacylate (EGDMA). In an embodiment, themonomer is hydroxylethyl acrylate (HEA).

In different embodiments, the polymer precursor is a photopolymerizablemonomer capable of forming a fluorescent polymer, a magnetic polymer, aradioactive polymer or an electrically conducting polymer. In anembodiment, the polymer precursor is water soluble. In an embodiment,the polymer precursor is a photopolymerizable fluorescent methacrylatemonomer. When the polymer precursor is fluorescent, the fluorophore mayabsorb the light used in the photopolymerization process. To compensate,the exposure time of the polymer precursor to the light and/or the lightintensity can be adjusted. In another embodiment, the polymer precursorneed not be capable of forming a fluorescent, magnetic, radioactive orelectrically conducting polymer if sufficient quantities of the polymercan be formed. In this embodiment, the polymer precursor can be anyphotopolymerizable polymer precursor or monomer. In an embodiment, thepolymer precursor can be an acrylate or a mixture of acrylates. Thepolymer precursor can also comprise a chromophore. In an embodiment, thephotoinitiator and chromophore preferentially absorb differentwavelengths of light.

In another embodiment, the polymer precursor solution further comprisesmicroparticles or nanoparticles that can be used to trigger a measurableresponse and these particles are incorporated into the polymer massduring polymerization. For example, the particles may be fluorescent,magnetic, radioactive, electrically conducting, or absorptive/colored.As used herein, nanoparticles have an average size greater than or equalto 1 nm and less than 1000 nm. As used herein microparticles have anaverage size greater than or equal to 1 micron to less than 1000microns. In an embodiment, the particles are nanoparticles. In differentembodiment, the average size of the nanoparticles is from 1 to 500 nm,from 5 to 200 nm, from 5 to 100 nm, from 10 to 50 nm, or from 20 to 40nm.

In an embodiment, the particles are fluorescent particles. Fluorescentparticles known to the art include fluorescently labeled microspheresand nanospheres. These particles include surface labeled spheres,spheres labeled throughout, and spheres possessing at least one internalfluorescent spherical zone (as described in U.S. Pat. No. 5,786,219 toZhang et al.) Other fluorescent particles known to the art includequantum dots (QDots). These include naturally fluorescent cadmiumselenium nanoparticles that have optical properties that are tunablewith their size.

In an embodiment of the present invention, the fluorescent particles aremicrospheres or nanospheres having fluorescent dye substantiallycontained within the particle, rather than being present only on thesurface of the particle. Such particles may also be referred to ashaving the dye encapsulated within the particles. Such particles arecommercially available and are commonly termed microspheres (even forparticle diameters less than one micrometer). Containment of the dyewithin the beads is believed to limit interaction of the dye with theother components of the polymer precursor solution, since physicalcontact of the dye with these components is limited. In an embodiment,the microspheres or nanospheres are polystyrene particles or beads. Thesurface of the microspheres or nanospheres may be modified in a varietyof ways. Commercially available modifications includecarboxylate-modified products, amine-modified products, sulfate andaldehyde-sulfate modified products. In an embodiment, nanospheres usedin the present invention are carboxylate modified; the resultingmicrospheres has been stated to be highly charged and relativelyhydrophilic (Molecular Probes Product Information, “Working withFluoSpheres® Fluorescent Microspheres, 1994).

In an embodiment, the polymerization product is a polymer gel and theparticles are incorporated into the gel. In an embodiment, the networkstructure of the gel allows encapsulation of the particles withoutcovalent attachment of the particles to the gel network. In anotherembodiment, the particles are covalently attached to the polymer formed.

Surface treatment of the particles may be used to obtain covalentattachment of the particles to the polymer formed. The appropriate formof surface treatment may vary with the type of particle and the type ofpolymer, but may include attachment of functional groups, monomers orpolymers to the surface. In an embodiment, pendant acrylic monomers maybe coupled to the surface of the particles. Acrylic monomers may becoupled to the particle surface by reaction of commercially availableparticles with acrylate molecules. Polymers may also be attached to thesurface through polymerization from the surface.

In an embodiment, the dye contained within particles is selected forcompatibility with the photopolymerization process. The absorptionspectrum of the dye may overlap that of the initiator, so long aspolymerization is not reduced to unacceptable levels. If the absorptionspectra overlap, the intensity and/or exposure of the light may beadjusted accordingly to compensate.

In another embodiment, the dye contained within the particles isselected for compatibility with a particular detection device. Forexample, the dye may be selected so that it has an emission maximumsuitable for a particular filter set.

In an embodiment, fluorescent particles suitable for use withfluorescein or fluoresciein-derivative initiators can encapsulate dyeshaving excitation and emission maxima which fall within a relativelybroad range of values. In an embodiment, the fluorescent particles canhave an absorption/excitation maximum which falls in the range fromapproximately 500 to approximately 670 nm and an emission maximum whichfalls in the range from approximately 510 to approximately 690 nm.Suitable fluorescent particles include, but are not limited to, Crimson(excitation/emission maxima of 625/645 nm), Nile Red (broadexcitation/emission bandwidths of 535/575 nm), Yellow-Green(excitation/emission maxima 505/515 nm), and Dark Red(excitation/emission maxima of 660/680) FluoSpheres®, all available fromInvitrogen.

In an embodiment, the amount of oxygen dissolved in the polymerprecursor solution is minimized to minimize oxygen inhibition of thepolymerization process. In an embodiment, the oxygen content of thesolution is less than about 1×10⁻⁵ moles/liter. The amount of oxygendissolved in the solution may be minimized by control of the atmosphereunder which polymerization takes place, reducing the oxygen content ofthe polymer precursor solution by flowing a gas through it and/or theaddition of oxygen inhibition agents. The “purge” gas used to reduce theoxygen content of the polymer precursor solution may be flowed throughthe polymer precursor solution prior to contact of the precursorsolution with the target probe complex, during contact of the precursorsolution with the target probe complex, and/or during polymerization. Inan embodiment, oxygen inhibition agents such as multifunctional thiolreagents are not used. In an embodiment, the oxygen content of thepolymer precursor solution during polymerization can be minimized byperforming the polymerization in an enclosure and introducing a gaswhich does not have a substantial oxygen content into the enclosure. Indifferent embodiment, the oxygen content of the gas is less than about10%, less than about 5% and less than about 1%. Suitable gases include,but are not limited to, commercial purity argon and nitrogen. Theatmosphere in the enclosure may be obtained by simply filling theenclosure with the desired gas, or by flowing gas through the enclosure.The enclosure can also be evacuated and backfilled with gas. The oxygencontent of the polymer precursor solution can also be reduced prior topolymerization by bubbling a suitable gas through the solution, or byany other method known in the art. Suitable gases include those which donot have a substantial oxygen content, such as argon and nitrogen.Oxygen and air are not suitable purge gases.

The solution may further comprise oxygen inhibition agents and/orcross-linking agents. In an embodiment, the oxygen inhibition agent is amultithiol (Bhanu, V. A. & Kishore, K. (1991) Role of Oxygen inPolymerization Reactions, Chemical Reviews 91: 99-117). The amount ofoxygen inhibition agent should not be so much that polymerization occursin the bulk of the solution rather than from the surface. However,oxygen inhibition agents which can act as chain transfer agents are notrecommended for use with radical polymerization processes when it isdesired to form sufficient quantities of the polymer for visualdetection. A crosslinking agent can stabilize the polymer that is formedand improve the amplification factor (Hacioglu B., Berchtold K. A.,Lovell L. G., Nie J., & Bowman C. N. (2002) Polymerization Kinetics ofHEMA/DEGDMA: using Changes in initiation and Chain Transfer Rates toExplore the Effects of Chain-Length-Dependent Termination. Biomaterials23:4057-4064). Finally, a small amount of inhibitor can be added to theformulation to limit background polymerization caused by impurities andtrace radicals formed by absorption by molecules other than theinitiator.

The photoinitiator-labeled target-probe complex and polymer precursorare exposed to light, thereby forming a polymer. Photopolymerizationoccurs when polymer precursor solutions are exposed to light ofsufficient power and of a wavelength capable of initiatingpolymerization. The wavelengths and power of light useful to initiatepolymerization depends on the initiator used. Light used in theinvention includes any wavelength and power capable of initiatingpolymerization. Preferred wavelengths of light include ultraviolet orvisible. In different embodiments, the light source primarily provideslight having a wavelength between 200 and 400 nm, between 200 nm and 380nm, or from 200 nm. In an embodiment, the light source primarilyprovides light having a wavelength between 400 and 700 nm. Any suitablesource may be used, including laser sources. The source may be broadbandor narrowband, or a combination. The light source may provide continuousor pulsed light during the process. Both the length of time the systemis exposed to UV light and the intensity of the UV light can be variedto determine the ideal reaction conditions. For fluorescence detection,the exposure time and light intensity can be varied to obtain maximalfluorescence signal from spots on a microarray and minimal fluorescencesignal from the background. In an embodiment, the intensity of UVradiation is selected so that an appropriate dose of UV radiation can bedelivered in less than about one-half hour.

In an embodiment, after polymerization, unpolymerized polymer precursoris removed. The unpolymerized polymer precursor can be removed byrinsing, for example by rinsing with water or an aqueous solution. Theunpolymerized polymer precursor need not be removed if formation of thepolymer is to be detected by its refractive index or by other means thatwould not be interfered with by the presence of the unpolymerizedpolymer precursors.

If the hydrogel polymer is not substantially fluorescent, magnetic,radioactive, or electrically conducting, the hydrogel can be contactedwith a detectable solution which is fluorescent, magnetic, radioactive,or electrically conducting so that the hydrogel absorbs a sufficientquantity of the detectable solution. After the detectable solution isabsorbed into the hydrogel, the excess solution is removed beforedetection.

In an embodiment, the polymer formed is detected by fluorescence,magnetic, radioactive or electrical detection methods as are known tothe art. If the probes are part of a DNA microarray, a commerciallyavailable microarray scanner and/or imager can be used to detect polymerformation. DNA microarray scanners and/or imagers are commerciallyavailable that can detect fluorescent or radioisotopic labels.

In another embodiment, sufficient quantities of the polymer are formedthat polymerization can be detected by visual inspection. Polymerizationwhich is detectable by visual inspection may also be detectable viaimage analysis of photographs or digital images of part or all of thearray or substrate. Polymerization can be detected by visual inspectionwhen there is sufficient contrast between the areas where polymer hasformed and the unpolymerized monomer, the other areas of the array orthe array substrate. In an embodiment, the areas where the polymer hasformed appear to be a different color (or shade of gray) than theunpolymerized monomer, the other areas of the array or the arraysubstrate. For example, after unpolymerized monomer is removed, theareas where polymer has formed may appear darker than the arraysubstrate. In another embodiment, the areas where the polymer has formedcan have a different transparency than the unpolymerized polymerprecursor. For example, the unpolymerized polymer precursor may be clearand the polymer more opaque and whitish in color. The amount of polymerrequired for visual detection of polymer formation may depend upon thepolymer. For acrylate mixtures such as mixtures of Hydroxyethyl Acrylate(HEA) and Ethylene Glycol Dimethacrylate (EGDMA), the thickness of thepolymer formed can be greater than about 1 micron, or greater than about5 microns. Determination of polymer formation may be made with eitherswollen or dried gels. For accurate polymer film thickness measurements,the gels are typically dried.

Analysis of polymer formation can allow identification of the target.Methods of design and analysis of DNA microarrays for identification oftarget molecules are known to the art (Vernet, G. (2002) “DNA-ChipTechnology and Infectious Diseases” Virus Research 82:65-71). Similarmethods, appropriately modified, can be applied to other types ofmicroarrays and molecular recognition events.

The sensitivity of the detection methods of the invention can bemeasured in several ways. In an embodiment, a microarray dilution chipmay be prepared having spots with differing amounts of a target or testspecies or molecule which is capable of binding with the photoinitiatorlabel. In an embodiment, a test species is chosen which is expected tohave similar binding/capture properties for the photoinitiator label. Indifferent embodiments, the test species may be a biotinylatedoligonucletide or double stranded DNA hybrid. The photoinitiator labelis then attached to the target or test species. Afterphotopolymerization, it may be observed which spots on the chip resultin a detectable amount of polymer formation. When the surfaceconcentration of the target or test species of a given spot is known,polymer formation at the spot indicates detection of at least thatconcentration level of the target or test species. When the size of thespot is known, the sensitivity can be determined in terms of the numberof molecules required for detection. The sensitivity of the methoddetermined by detection of a test species is expected to be related tothe actual sensitivity of the method for detection of a target species,but may also be affected by factors such as labeling efficiency andhybridization efficiency. The assessed sensitivity of the method maydepend on the sensitivity of method used to assess whetherpolymerization has occurred, with more sensitive polymerizationassessment methods demonstrating greater sensitivity of the detectionmethod. For example, when polymerization is assessed via opticalobservation the observed sensitivity may be lower then whenpolymerization is assessed via profilometry measurements. In anembodiment, the methods of the invention are capable of detecting aconcentration of 0.4 attomoles (4×10⁻¹⁹ moles) when detection isassessed optically (by eye or with an optical microscope) and 0.1attomoles when detection is assessed through profilometer measurements.

An amplification factor, which can be defined as the number ofpropagation reactions occurring per molecular recognition event, can becalculated from analysis of the thickness of the polymer films. Thevolume of the film may be divided by the density of the polymer toobtain the mass of polymer formed. Division by the molecular weight ofthe monomer/polymer precursor/repeat unit gives the number of monomersreacted into the polymer matrix. Dividing the number of monomers permicron squared by the number of molecular recognition events per micronsquared gives the amplification factor. In an embodiment, theamplification factor of at least 10⁶ or 10⁷ is obtained for eachmolecular recognition event. If the amplification factor is determinedacross a given concentration range, the thickness of the film can beused as a measure of the number of molecular recognition events (in thatconcentration range).

In an embodiment, the number of molecular recognition events can bequantitatively determined by comparison of the observed film thickness(or other detectable characteristic) to a reference calibration curve offilm thickness (or other detectable characteristic) versus target ortest species concentration on a solid surface. The reference calibrationcurve or correlation is obtained for similar photopolymerizationconditions (photoinitiator label, polymer precursor solution, lightsource, light intensity, and light exposure time). This calibrationcurve may be obtained by preparation of a dilution chip fabricated byspotting decreasing concentrations of the target or test molecules ontoa substrate, then attaching photoinitiator labels to the target or testmolecules and photopolymerizing a monomer solution from the surfacebound initiators according to the detection methods of the invention.The concentrations of the target or test molecules can be determined fora similar dilution chip by attaching a fluorescent label to themolecules and then characterizing the surface with fluorescent detectionmethods.

The relationship between the detectable characteristic and the surfaceconcentration may be useful over a limited range of target or testspecies surface concentrations. This useful range may also be referredto as the dynamic range. The lower end of the range may be limited bythe detection level of the detectable characteristic. The upper end ofthe range may be limited by a “saturation” effect. For example, in somecases the thickness of the polymer film appears to increase much moreslowly at higher surface concentrations. Measurements in the usefulrange may be obtained by repetition of the experiment with differentexperimental conditions. In another embodiment, the probe array may beprovided with spots having different concentrations of the same probemolecule to increase the likelihood that at least one spot will have asurface concentration in the useful range.

In another embodiment, quantitative analysis of the target moleculeconcentration can be performed by changing the exposure timesystematically across an array of sample spots. Comparison of the doseat which polymerization is observed with a calibration curve of doseversus concentration allows quantitative determination of theconcentration. In another embodiment, the probe array may be providedwith different concentrations of the same probe molecule and a singleexposure time used for the array.

In different embodiments, the methods of the invention are capable ofdetecting as few as 10³ or ˜10⁴ labeled oligonucleotides using minimalinstrumentation, such as an optical microscope or CCD camera. In otherembodiments, the methods of the invention are capable of visualdetection of concentrations as few as 100, 50, 25, 10, 5, 1, 0.5, 0.1 or0.005 biomolecules/μm².

As used herein, “comprising” is synonymous with “including,”“containing,” or “characterized by,” and is inclusive or open-ended anddoes not exclude additional, unrecited elements or method steps. As usedherein, “consisting of” excludes any element, step, or ingredient notspecified in the claim element. As used herein, “consisting essentiallyof” does not exclude materials or steps that do not materially affectthe basic and novel characteristics of the claim. Any recitation hereinof the term “comprising”, particularly in a description of components ofa composition or in a description of elements of a device, is understoodto encompass those compositions and methods consisting essentially ofand consisting of the recited components or elements. The inventionillustratively described herein suitably may be practiced in the absenceof any element or elements, limitation or limitations which is notspecifically disclosed herein.

The terms and expressions which have been employed are used as terms ofdescription and not of limitation, and there is no intention in the useof such terms and expressions of excluding any equivalents of thefeatures shown and described or portions thereof, but it is recognizedthat various modifications are possible within the scope of theinvention claimed. Thus, it should be understood that although thepresent invention has been specifically disclosed by preferredembodiments and optional features, modification and variation of theconcepts herein disclosed may be resorted to by those skilled in theart, and that such modifications and variations are considered to bewithin the scope of this invention as defined by the appended claims.Whenever a range is given in the specification, all intermediate rangesand subranges, as well as all individual values included in the rangesgiven are intended to be included in the disclosure. When a Markushgroup or other grouping is used herein, all individual members of thegroup and all combinations and subcombinations possible of the group areintended to be individually included in the disclosure.

In general the terms and phrases used herein have their art-recognizedmeaning, which can be found by reference to standard texts, journalreferences and contexts known to those skilled in the art. The abovedefinitions are provided to clarify their specific use in the context ofthe invention.

All patents and publications mentioned in the specification areindicative of the levels of skill of those skilled in the art to whichthe invention pertains.

All references cited herein are hereby incorporated by reference to theextent that there is no inconsistency with the disclosure of thisspecification. Some references provided herein are incorporated byreference herein to provide details concerning additional startingmaterials, additional methods of synthesis, additional methods ofanalysis and additional uses of the invention.

Example 1 Synthesis of a Water Soluble Initiator

As shown in Scheme 1, synthesis of a water soluble photoinitiator(preferred for compatibility with a BioChip) was achieved by startingwith commercially available Irgacure 2959 (left most structure, CibaSpecialty Chemicals (http://www.cibasc.com)). Irgacure 2959 wasdissolved/suspended in chloroform along with succinic anhydride and acatalytic amount of 4-dimethylaminopyridine. The solution was refluxed,with stirring, for 12 hours at 65° C. In both chloroform and water, theproduct was soluble while the starting materials were sparingly soluble.The product structure was verified by NMR and shown to function as aphotoinitiator by monitoring the double bond conversion of an acrylatemonomer using time-resolved FTIR.

Example 2 Functionalization of Avidin with Photoinitiator

Avidin is often labeled with dye molecules by modification of its manylysine residues (for example, Pierce Biotechnology sells a kit for thispurpose). These types of modifications do not disrupt avidin's abilityto bind to biotin (Wilbur, D. S.; Hamlin, D. K.; Buhler, K. R.; Pathare,P. M.; Vessella, R. L.; Stayton, P. S.; To, R. (1998) “Streptavidin inantibody pretargeting. 2. Evaluation of methods for decreasinglocalization of streptavidin to kidney while retaining its tumor bindingcapacity” Bioconjugate Chemistry 9: 322-330). Here, as shown in Scheme2, the lysine residues have been modified with a photoinitiator (ratherthan a dye) by coupling the carboxylic acid functional group of thephotoinitiator to the amine of the lysine residue. The result isformation of a peptide bond between the initiator and the protein.Avidin and an excess of the initiator (the product in Scheme 1,represented by In in Scheme 2) were dissolved in an acid aqueousbuffered solution in the presence of the water-soluble coupling agent1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) (Hermanson, G. T.(1996) Bioconjuqate Techniques San Diego, Calif.: Academic Press. p.435). The reaction proceeded at room temperature for 12 hours, and theproduct was collected by ultracentrifugation through a 3,000 MW cutofffilter. Biotin binding capabilities were verified using the HABA assay(Wilcheck, M., (b) Bayer, E. A. Eds. (1990) “Protein biotinylation”Methods in Enzymology 184: 138-160).

Example 3 Synthesis of a Polymer Labeled with Photoinitiator andStreptavidin

Scheme 3 illustrates the formation of a polymer labeled with Irgacure2959 photoinitiator (denoted by I) and streptavidin (denoted by

). Macroinitiators were synthesized using poly(acrylicacid-co-acrylamide) (MW=200,000 g/mol), Irgacure 2959, and streptavidinas starting materials. The water-soluble coupling agent1-ethyl-3-[3-dimethylaminopropyl]carbodiimide (EDC) and theintermediate-stabilizing molecule N-hydroxsuccinimide (NHS) were used tocreate amide linkages between some of the acrylic acid subunits andstreptavidin and to create ester linkages between other acrylic acidsubunits and the initiator Irgacure 2959 (12959). Still other acrylicacid subunits were left unmodified to assure the water solubility of theresulting macroinitiator. Two types of reaction conditions were variedin order to change the number of initiators that coupled per chain.First, the stoichiometry of reactants was varied up to the limit ofsolubility of the initiator in the aqueous conjugation buffer. Second,the length of time allowed for the activation step (in which EDC and NHSreact with the carboxylic acid subunits of poly(acrylicacid-co-acrylamide)) was varied. Table 1 summarizes results from threesets of reaction conditions.

TABLE 1 Acti- Initiators vation Absorbance per Stoichiometry time at 300chain* 1 685 μL of 1 mg/ml I2959 10 0.8 140 440 μL of 1 mg/ml EDCminutes 260 μL of 1 mg/ml NHS 2 685 μL of 1 mg/ml I2959 15 1.3 40 440 μLof 1 mg/ml EDC minutes 260 μL of 1 mg/ml NHS  3** 68.5 μL of 1 mg/mlI2959 15 0.5 88 44 μL of 1 mg/ml EDC minutes 26 μL of 1 mg/ml NHS*Calculated using a standard curve of absorbance at 300 of the initiatoras a function of initiator concentration. **Reaction 3 was brought up toequal volume with Reactions 1 and 2 using the conjugation buffer.

After activation, the initiator and protein are added and the reactiontakes place in the presence of EDC and N-hydroxysuccinimide (NHS) atroom temperature for about two hours.

Example 4 Macroinitiator Synthesis

In the event that a label sequence containing a single initiator doesnot provide a high enough level of amplification with the shortirradiation times necessary to minimize background fluorescence,macroinitiators, in which many initiators are present on a singlemolecule, can be used. Synthesis of a macroinitiator can be achievedthrough a living (or controlled) radical polymerization method prior toutilization on the microarrays (Kamigaito M., Ando T., & Sawamoto M.(2001) “Metal-Catalyzed Living Radical polymerization. Chemical Reviews101: 3689-3746). Atom transfer radical polymerization (ATRP) schemes canbe used to control the macroinitiator molecular weight, composition andarchitecture (block copolymers, branching, etc.) (Matyjaszewski, K. &Jianhui Xia, J. (2001) Atom Transfer Radical Polymerization ChemicalReviews 101: 2921-2990).

Macroinitiator synthesis can be performed, as presented in Scheme 4, bystarting with an oligonucleotide terminated in an amine group. The amineterminus is functionalized with an ATRP initiator, which initiatespolymerization of the desired compounds. As many as three differentmonomers (an initiator, a spacer, and a photosensitizer) may need to becopolymerized. If necessary to improve hybridization to the probeoligonucleotide, a spacer molecule (for example (poly ethylene glycolmethacrylate)) is used between the initiating block of themacroinitiator and the oligonucleotide part of the label sequence. Thespacer may also be used for controlling solubility of the macroinitiator(by changing the hydrophobicity/hydrophilicity) and the macroinitiatormolecular weight. Photosensitizing components are incorporated into themacroinitiator in systems where minimization of the backgroundpolymerization is necessary.

Example 5 Synthesis and Photopolymerization of a Fluorescent Monomer

The N-hydroxy succinimide ester of fluorescein was purchased from PierceBiotechnology (Rockford, Ill.) and 2 mg were dissolved in 100 μL of DMSOwhile 0.5 mg of 2-aminoethyl methacrylate was dissolved in 900 μL ofsodium bicarbonate buffer, pH 8.2. The two solutions were combined andplaced on a shaker for two hours. The solvent was lyophilized off. Thestructures of the fluorescent monomer and the polymer that results fromirradiating the monomer with UV light were verified by NMR.

The monomer can be polymerized by irradiating with 365 nm ultravioletlight for one minute.

Example 6 Formation of a Hydrogel from a Polymer Labeled withPhotoinitiator and Streptavidin and Detection of the Hydrogel Formed

The polymer of Example 3 was reacted with a microarray having biotincovalently bound to the microarray substrate. A hydroxylethyl acrylatemonomer solution was placed in contact with the array by pipetting thesolution into a HybriWell (Grace Biolabs) which covers the array. Sixtyμl of an aqueous solution was used which contained hydroxylethylacrylate, initiator (product of Scheme 1 and Scheme 3), cross-linkingagent (ethylene glycol dimethacrylate, 3% by volume), and oxygeninhibition agent (mercaptoethanol, 5×10⁻⁴% by volume).

The monomer was then photopolymerized to form a hydrogel by irradiatingthe array with 365 nm light for about 1 minute.

After polymerization, the microarray was rinsed with water and then asolution containing Rhodamine B, concentration 100 nM, was pipettedbetween a glass coverslip and the microarray. The microarray was exposedto the fluorophore containing solution for about 30 minutes. Themicroarray was then rinsed with water to remove excess fluorophore.

FIG. 3A is an image of the array after polymerization. The grid ofreacted biotin spots (100) are bright and indicate areas of formation ofhydrogel swollen with the fluorescent solution. The detector used was anAgilent Microarray Scanner (Fluorescence Ave: 2600, Std: 1200Background: 1200Signal to Noise: 2.2). FIG. 3B shows negative controlsspots (110) on the same array (Fluorescence Ave: 900, Std: 30,Background: 800, Signal to Noise: 1.1)

Example 7 On-Chip Hybridization, Amplification, and Detection on a FluChip

On-chip signal amplification by photopolymerization was tested on acustom microarray designed to detect and subtype the influenza virus. Asshown in FIG. 4, the chip had a column of positive control spots (80),spots to capture RNA of influenza C (91), spots to capture RNA ofinfluenza B (93), and spots to capture RNA of influenza A (95). Therewere three spots in each column. The sequence that is complementary tothe positive control spots (but not to the other nine spots on thearray) was purchased from Qiagen (Valencia, Calif.) with a 5′ biotinmodification. Published protocols were used to hybridize a 1 μM solutionof this oligonucleotide to the microarray. The hybridization procedurewas as follows:

Pre-Hybridization.

-   -   1. Boil dH₂O.    -   2. While dH₂O is boiling, wash microarray in 0.1% sodium dodecyl        sulfate (SDS) for 2-10 min on rocker. (0.5 ml 10% SDS in 50 ml        total volume).    -   3. Transfer to 2× standard saline citrate (SSC) for 2 min. (5 ml        20× in 50 ml total volume).    -   4. Immerse slide in boiling/hot (>95° C.) water for 2-3 min.    -   5. Remove slide, blot on kimwipe.    -   6. Immerse slide in ice-cold ethanol bath for 2-5 min. Store        EtOH at −20° C.    -   7. Remove slide, blot on kimwipe.    -   8. Centrifuge ethanol off of slide. (50 ml conical tube, 1000        rpm, 3 min).

Hybridization.

-   -   1. Pipette 10 μL of water or buffer into the humidification        slots, place microarray in hybridization cassette.    -   2. Hybridization solution:        -   10 μL 50 micromolar oligo soln (phosphate-buffered saline            (PBS) or tris(hydroxymethyl) aminomethane (Tris))        -   10 μL 10×SSC        -   5 μL 1% Tween 20 (polyoxyethylene-20-sorbitan monolaurate,            ICI)        -   5 μL 10        -   mM MgCl₂        -   20 μL H₂O    -   3. Pipette 7 μL of this solution between a coverslip covering        the microarray and the slide.    -   4. Seal cassette, submerge in water bath (40-45° C.) for at        least 1.5-2 hrs.

Washing.

-   -   1. Transfer microarray immediately into 1×SSC (2.5 ml 20× in 50        ml total)/0.1% SDS (0.5 ml 10% SDS in 50 ml total). 5 min.    -   2. Transfer microarray into 0.1×SSC (0.25 ml 20× in 50 ml)/0.1%        SDS. 5 min.    -   3. Immerse briefly in 0.1×SSC to remove SDS.        Rinse with water, dry with N₂.

Following hybridization, 7 μL of a 1 mg/ml solution of BSA was pipettedbetween a glass coverslip and the microarray to block nonspecificbinding. Following twenty minutes of incubation in a humid hybridizationchamber, the array was rinsed with water. Subsequently, 7 μL of a 1mg/ml initiator-functionalized avidin solution was pipetted between aglass coverslip and the microarray. After twenty minutes of incubationand brief rinsing with water, 7 μL of a saturated solution of thefluorescent monomer in water was pipetted between the glass coverslipand the microarray. The array was irradiated with 365 nm UV light forone minute, rinsed with water, and imaged using an Agilent (Wilmington,Del.) microarray scanner. The excitation wavelength (λ_(ex)=532 nm) andcollection wavelength (centered at 575 nm) were not optimal for thefluorescein derivative (product in Scheme 3). FIG. 4 is the resultingimage

In FIG. 4, the lighter spots represent detected fluorescence. Though thebackground fluorescence remains relatively high despite the BSAtreatment, the positive control spots (80) are definitely morefluorescent than the spots designed to capture the genetic material ofinfluenza A, B and C (these spots effectively served as three differentnegative controls in this experiment). The faint gray vertical lines inFIG. 4 are a well understood artifact. Glass slides were placed betweenthe microarray and the UV light source to block high frequency UV lightthat is harmful to the fluorophore. The vertical lines arise from theseam between two glass slides that were used as a filter in this manner.

Two other control experiments were performed. Without the BSAnonspecific binding blocking step, and all other steps held constant,the entire image yielded significant fluorescence, indicatingnonspecific binding of the initiator. In addition, without the additionof avidin-functionalized initiator, when all other steps remainedidentical, none of the spots exhibited fluorescence yet the backgroundwas still high. These results suggest that the fluorescent spots in FIG.4 do indeed result from an interaction between the monomer and theselectively bound initiator. These tests, taken together, indicate thatphotopolymerization is clearly a viable means to obtain selective signalamplification directly on a DNA microarray.

Without wishing to be bound by any specific theory, one possible causeof nonspecific binding is reaction of the fluorescent monomer with thealdehyde coating on the glass. To eliminate this source of nonspecificbinding, alternative attachment chemistries are available, or theunreacted aldehydes outside of the oligonucleotide spots can bepassivated or reduced. If macroinitiators containing larger numbers ofinitiators are used, the irradiation time can be reduced. To furtherreduce nonspecific binding, an inhibitor can be included. This techniquewill be effective if the number of initiation sites inside thehybridized spots (specific, due to the presence of many initiators) ismuch greater than the number of possible sites outside the spots(nonspecific, absorption and initiation occurring without initiator, andnon-specific binding). False positives can be minimized by the use ofmacroinitiators, photosensitizers/inhibitors and optimal initiation timeand light intensity, combined with judicious choice of probe and labelsequence and steps to prevent non-specific binding of the initiator tothe array

Example 8 Quantification of Number of Fluors

The number of fluorescent molecules incorporated into the polymer andtherefore bound to the microarray surface can be quantified. One test isto measure the shape and size of the resulting spot after polymerizationis complete using either atomic force microscopy or profilometry. Withthis information and knowledge of the mass fraction of fluor within themixture and the polymer density, it is possible to calculate relativelyaccurately the number of fluors within the polymer. This approachminimizes error due to any fluorescence quenching within the polymer.Quenching is quantified and minimized by conducting a study offluorescence intensity as a function of mass percent fluor in thepolymer. Due to the amorphous nature of the polymer, quenching is notexpected to be significant.

Example 9 Recognition Between Biotin Arrays and Polymers Functionalizedwith Streptavidin and Photoinitiator

A biotin array was contacted with a macroinitiator solution comprising apoly (acrylic acid-co-acrylamide) polymer backbone (MW=200,000)functionalized with streptavidin and Irgacure 2959. The reactionconditions for forming the macroinitiator were as given in Table 1. Thearray was then washed to remove macroinitiator not attached to thebiotin array. The photoinitiator-labeled array was then contacted with amonomer solution comprising Hydroxyethyl Acrylate (HEA) and EthyleneGlycol Dimethacrylate (EGDMA). The monomer solution had been previouslypurged with argon to reduce its oxygen content. The photoinitiatorlabeled array and monomer solution were then exposed to 5 mW/cm², 365 nmUV light for 20 minutes in an argon atmosphere. Unpolymerized monomersolution was then removed by washing. Details of biotin arraypreparation, monomer preparation, recognition and amplificationprocedures are given below.

The product of reaction 1 (see Table 1) led to the formation of polymerspots that were up to 10 microns thick, as measured with a profilometer.FIG. 5 compares a profilometer trace across the hydrogel spots with acontrol profilometer trace. A digital image of the polymer spots showsthat they are darker than the surrounding substrate. The product ofreaction 2 led to polymerization everywhere (not just within the spots).The product of reaction 3 led to the formation of polymer spots thatwere up to 0.1 micron thick, as measured using a profilometer.

Biotin Array Preparation:

-   -   1. Vapor-deposit aminopropyltriethoxysilane onto a piranha        cleaned silicon (or glass) substrate. Piranha cleaning involves        placing substrates in 70% v/v sulfuric acid, 30% v/v 30%        hydrogen peroxide at 90° C. for 1 hour. Vapor deposition        includes placing substrates into a purged Teflon container along        with an open vial containing the silane for two hours at 90° C.    -   2. Spot 1 mg/mL biotin-polyethylene glycol (PEG)-benzophenone in        1×PBS on an amine substrate. Set relative humidity in spotter to        75%. Allow substrates to dry in spotter for half hour after        spotting is complete and then move to ambient conditions to dry        for at least 2 hrs.    -   3. Covalent attachment of biotin to the surface: irradiate the        substrate with UV light at 5 mW/cm2 using Black Ray lamp at 365        nm for 10 minutes.    -   4. Blocking of nonspecific binding: soak arrays in 1 mg/mL dry        milk in water solution for two hours while slowly agitating with        shaker.    -   5. Wash arrays ×3 in water for 5 minutes, then dry the slides        using a stream of nitrogen.

Monomer Preparation:

-   -   1. Hydroxyethyl Acrylate (HEA) and Ethylene Glycol        Dimethacrylate. (EGDMA) are deinhibited from MEHQ by three        consecutive distillations.    -   2. Make 300 μL of 97 vol % HEA and 3 vol % EGDMA.    -   3. Purge monomer of dissolved oxygen by bubbling argon through        the monomer for 10 minutes and then seal the container with        Parafilm® when done.

Recognition:

-   -   1. Pipette a 20 μL of macrophotoinitiator solution (1.4 mg poly        (acrylic acid-co-acrylamide backbone per ml in 0.1 M        2-(N-morpholino) ethane sulfonic acid (MES) buffer, 0.5 M NaCl,        pH 5) directly onto array and then drop a plastic coverslip onto        drop, making sure drop spreads uniformly over coverslip area.        Place slide in humid chamber for 20 minutes.    -   2. Wash slide ×3 in water for 5 minutes, do not dry slide with        nitrogen.

Amplification:

-   -   1. Place silicone isolator around spots, making sure it adheres        to the plate well. This will keep the monomer from spreading        everywhere on the substrate.    -   2. Place plate in argon chamber and then pipette 300 μL of        monomer inside the well formed by the isolator and the        substrate.    -   3. Put glass top on chamber, turn argon on, let it purge for 5        minutes. After 5 minutes, tighten down the top on the chamber.    -   4. Irradiate the plate in the chamber for 20 minutes under 5        mW/cm², 365 nm UV light.    -   5. Remove plate from purge chamber and wash ×3 in water, dry        with nitrogen. Look for polymer spots that have grown from the        array.

Example 10 Polymerization of a Chromophore-Containing Monomer Using aMacroinitiator Incorporating Tertiary Amines

A chromophore-containing monomer is polymerized using a photoinitiatorwhich preferentially absorbs light at a different wavelength than thechromophore. A chromophore which preferentially absorbs UV light can bepaired with a photoinitiator which preferentially absorbs visible light.Scheme 6 illustrates formation of an acrylate monomer incorporating thechromophore Cascade Blue Ethylene Diamine, which preferentially absorbslight at approximately 400 nm. Scheme 7 illustrates formation of amacroinitiator comprising a polymer incorporating multiple tertiaryamines. The photoinitiator for the polymerization of the monomercomprising the tertiary amine and the acrylate group with acrylic acidis (for water solubility and to provide a functional group thatstreptavidin can be coupled to) Irgacure 184. 1-hydroxycyclohexyl phenylketone (Ciba-Geigy). The polymer chain is coupled to at least onemolecular recognition group, shown as streptavidin in Scheme 7. As shownin Scheme 8, the tertiary amines of the macroinitiator and CQ form atwo-part initiator system which most strongly absorbs light at 469 nm.The radical species shown in Scheme 8 propagates through thecarbon-carbon double bonds of the chromogenic monomer that is theproduct of Scheme 6 to form a chromogenic monomer.

Example 11 Recognition Between Biotinylated Oligonucleotide Arrays andPolymers Functionalized with Neutravidin and Photoinitiator

Dual-functional macrophotoinitiators were synthesized by couplingwater-soluble photoinitiators and Neutravidin to a fraction of thecarboxylate residues of a high-molecular weight copolymer of acrylicacid and acrylamide (Scheme 9) using aqueous carbodiimide couplingchemistry (Staros, J. V., Wright, R. W., Swingle, D. M. Enhancement byN-Hydroxysulfosuccinimide of Water-Soluble Carbodiimide-MediatedCoupling Reactions. Anal. Biochem. 156, 220-222 (1986)). UV absorbancemeasurements showed that each macroinitiator contained an average of 140initiators per chain, and HABA assays (Green, N. M. A spectrophotometricassay for avidin and biotin based on binding of dyes by avidin. Biochem.J. 94, 23c-24c (1965)) revealed 1-2 pendant Neutravidins per chain withretention of biotin-binding capability.

The ability of the macrophotoinitiator to recognize biotin-labeledoligonucleotides and to initiate polymerization of water-solublemonomers was tested on thin film biosensors (Jenison, R.; La, H.;Haeberli, A.; Ostroff, R.; Polisky, B. Clinical Chemistry 2001, 47,1894-1900; Jenison, R., Yang, S., Haeberli, A., Polisky, B.Interference-based detection of nucleic acid targets on optically coatedsilicon. Nature Biotechnol. 19, 62-65 (2001); Zhong, X. et al.,Single-nucleotide polymorphism genotyping on optical thin-film biosensorchips. Proc. Natl. Acad. Sci. U.S.A. 100, 11559-11564 (2003)). Thesesurfaces, because of a specifically designed optical interference layerin the substrate, were ideally suited to testing and optimizingpolymerization conditions since films as thin as 5 nm result in aneasily observable color change of the surface from gold to blue(Jenison, R.; La, H.; Haeberli, A.; Ostroff, R.; Polisky, B. ClinicalChemistry 2001, 47, 1894-1900). Further, the color of the film is adirect measure of the thickness of the film, and hence, a quantitativemeasure of the amount of polymer that is formed.

A 2×2 oligonucleotide array was spotted on thin film biosensors. Thespots in the first column contained biotinylated oligos (5 femtomoles inthe upper spot, and 0.5 femtomoles in the lower spot), while the spotsin the second column contain unlabeled oligos. Following a 10 minutedose of 5 mW/cm², 365 nm light, polymer grew only from the two spotscontaining biotinlyated oligonucleotides (5 fmoles and 0.5 fmoles) anddid not grow from the two spots containing unlabeled oligonucleotides.The polymer spots rapidly exceeded 100 nm in thickness, below which thesurfaces would yield quantitative information on the amount of polymer.Thus, the unique optical properties of the surfaces were not necessaryfor detecting a positive response as all polymer films beyond 100 nmappear white. The special optical properties of the surfaces were,however, useful for assessing the occurrence of bulk polymerization ornonspecific polymerization, as even small amounts of polymer would causea color change on the surface. Color changes were not observed,indicating the lack of both false negatives and bulk polymerization. Anegative control chip was subjected to identical conditions except for alack of exposure to the macrophotoinitiator. No polymerization resulted(at or above the visible limit of 5 nm), ruling out concerns that amolecule other than the macroinitiator initiated polymerization.

To determine a detection limit for surface-bound oligonucleotides, adilution chip was prepared with spots containing from femtomoles tozeptomoles of biotinylated oligos. A negative control spot containingonly unlabeled oligos was included on the chip. FIGS. 6 a-6 c show theresults obtained using photopolymerization for signal amplification. Asshown in FIG. 6 a, polymer spots were visible over the first six spotsin the array following a 10 minute incubation with avidin-functionalizedmacroinitiator and a 10 minute dose of 5 mW/cm², 365 nm light. Usingenzymatic amplification (HRP/TMB-dextran), only the first two spots werevisible. FIG. 6 b shows the maximum number of biotinylated oligospresent in each spot. The bottom right spot contained only unlabeledoligos and served as a negative control. FIG. 6 c shows the minimumradiation dose that was delivered to each spot prior to observation ofpolymer formation, where the dose is a product of the light intensityand exposure time. The error bars reflect variations in the intensityoutput from the lamp as measured with a radiometer.

The most dilute spot that is detected through the formation of a visibleamount of polymer contains on the order of 1000 biotinylatedoligonucleotides, orders of magnitude fewer than the numbers that aredetectable using enzymatic amplification methods. The picture shown inFIG. 6 a is representative of twenty trials; no false positives or falsenegatives (above the limit of detection) occurred.

Further, with constant light intensity exposure, spots containingvarying concentrations of the analyte do not appear simultaneously.Rather, as the surface-localized polymerization reactions progress,spots containing higher concentrations of biotinylated oligonucleotidesbecome visible before spots with lower concentrations of biotinylatedoligonucleotides become visible. FIG. 6 c shows the dose of light thatwas necessary to see each spot. This response of lower concentrationspolymerizing with larger irradiation doses was observed in adifferentiable manner across more than three orders of magnitude inanalyte concentration. This outcome provides a facile means forconverting qualitative detection schemes into a technique that isreadily able to quantify the amount of an analyte present at theselevels. Simply by changing the exposure time systematically across anarray of sample spots, for example by the movement of an opaque filmacross the surface, it is possible to quantify an analyte, even at theseextremely low levels. Thus, a simple change in exposure time across anarray can enable quantitative analysis of the target moleculeconcentration.

The macroscopic, visible response generated by this small number ofpossible recognition events is remarkable. Without wishing to be boundby any particular theory, we hypothesize that the large degree ofpolymerization is a result of high radical initiation rates occurringonly at the desired surface while radicals that propagate into the bulkdo not encounter radicals that lead to termination. The large degree ofpolymerization is believed to be further enhanced by the formation of acrosslinked, hydrogel polymer. Here, the monomer formulation wasoptimized to contain a crosslinking agent that hinders radicaltermination and facilitates the formation of extremely large amounts ofpolymer from each radical generated.

The relationship between the color of the spot and the thickness of thethin film within the spot has been reported previously; white spotscorrespond to film thicknesses of at least 100 nm (Jenison, NatureBiotech 2001). Using this thickness as a lower limit for the thicknessesof the polymer spots in FIG. 6 a, combined with knowledge of the numberof possible binding events and the density of the polymer, we calculatea minimum amplification factor of 10¹¹ monomers polymerized per bindingevent in the most dilute spot in FIG. 6 a. The density of biotinylatedbiomolecules in the last visible spot is ˜0.005 per μm²; this density isbelow the limit of detection of even the newest high-end fluorescencescanners, instruments that cost tens of thousands of dollars.

False positives are a substantial concern with any signal amplificationapproach to detection. Though we did not encounter problems with falsepositives in this study, the result shown in FIG. 6 c provides evidencethat should false positives arise with more complex samples orrecognition pairs that have less specificity than avidin and biotin, itwould be possible to shift the threshold of the positive response toexclude nonspecific interactions by selecting an appropriate dose oflight. Specificity of detection reagents is a limitation in manydiagnostic assays, including ELISAs. Usually, it is not the enzymaticamplification step that limits sensitivity in these assays, but rather,the antibody specificity. In this study, we compared the “top” enzymaticamplification step employed in many ELISA sandwich assays withpolymerization-based amplification. This comparison, in whichpolymerization provided an improvement in sensitivity by orders ofmagnitude. The exciting and rapidly progressing discoveries directedtoward improving the specificity of detection reagents (Jayasena, S. D.Aptamers: an emerging class of molecules that rival antibodies indiagnostics. Clin. Chem. 45, 1628-1650 (1999); Binz, H. K, Amstutz, P.,Pluckthun, A. Engineering novel binding proteins from nonimmunoglobulindomains. Nature Biotechnol. 23, 1257-1268 (2005); Brandt, O., Hoheisel,J. D. Peptide nucleic acids on microarray and other biosensors. Trendsin Biotechnology 22, 617-622 (2004); Liu, H., et al. A four-base pairedgenetic helix with expanded size. Science 302, 868-871 (2003); Boder, E.T., Midelfort, K. S., Wittrup, K. D. Directed evolution of antibodyfragments with monovalent femtomolar antigen binding affinity. Proc.Natl. Acad. Sci. 97, 10701-10705 (2000)) promise to make our findingsmore broadly applicable in the coming years.

Macrophotoinitiator synthesis and characterization Initiators andproteins were linked to the —COOH groups of a high molecular weightcopolymer of acrylic acid and acrylamide through ester linkages(initiators) and amide linkages (proteins). Though the acrylamidesubunits were not involved in the conjugation reaction, their presencewas important in the surface bound detection results shown in FIGS. 6a-6 c (macroinitiators made with an acrylic acid starting material inthe place of poly(acrylic acid-co-acrylamide) do not functionsimilarly). The ratio of acrylic acid to acrylamide can be approximately1:4 (n:m) Scheme 9 illustrates the synthesis.

A 1 mg/ml solution of N-hydroxy succinimide (NHS) (Aldrich) in 0.1 M MES(2-Morpholinoethanesulfonic acid) 0.5 M NaCl buffer, pH 6 and a 1 mg/mlsolution of the commercial initiator Irgacure 2959 (Ciba) in distilledwater were prepared and placed on a vortexer for 10 minutes until fullydissolved. 0.8 mg of poly (acrylic acid co-acrylamide) (200,000 MW,Aldrich) and 1 mg of the coupling agent1-Ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) (Aldrich) wereweighed out during this time. 260 μL of the NHS solution was pipettedinto the tube containing 0.8 mg of poly (acrylic acid co-acrylamide). 1ml of MES buffer was quickly added to the tube containing 1 mg EDC, and440 μL of this solution was quickly added to the tube containing NHS andpoly (acrylic acid co-acrylamide). This solution was placed on a shakeron a low setting for 15 minutes to allow time for activation of thecarboxylic acids of poly (acrylic acid co-acrylamide) by EDC and NHS.685 μL of the initiator solution and 50 μL of a 10 mg/ml Neutravidinsolution (Pierce, Neutravidin is a deglycosylated form of avidin) wereadded to the activated poly (acrylic acid co-acrylamide) solution, andthe reaction was allowed to proceed on a shaker on a low setting for onehour and forty-five minutes. At this time, the high molecular weightproduct was separated from unreacted smaller molecules using a 100,000molecular weight cutoff filter (Millipore) and a centrifuge. The spinrates and times recommended by Millipore were used. Purified productswere brought up to 500 μL total volume with MES buffer, and UV spectrawere collected. A calibration curve of initiator absorbance as afunction of concentration was made and used to determine the averagenumber of initiator substituents per macrophotoinitiator. HABA assayswere performed as described in Green et al. (Green, N. M. Aspectrophotometric assay for avidin and biotin based on binding of dyesby avidin. Biochem. J. 94, 23c-24c (1965).) to determine the averagenumber of Neutravidin substituents per macrophotoinitiator, and toverify retention of biotin binding capability. In our hands, thisreaction was very sensitive to any deviation from the above procedures.The non-standard stoichiometry of reactants described above was reachedempirically. Initial stoichiometry was 1× poly (acrylic acidco-acrylamide): 1000× photoinitiator: 1000×EDC: 1000×NHS: 1×Neutravidin; however, using this stoichiometry the resulting product wasa crosslinked hydrogel that was not useful. NMR was also used to verifythe product of the above reaction. Note regarding macroinitiator design:Commercial photoinitiators are commonly used as ˜1% by weight additivesin bulk polymerizations, a concentration many orders of magnitude higherthan could be expected in this new application, particularly near thedesired detection limit. Their UV extinction coefficients aresufficiently low that one-initiator-per-binding-event molecules wereunlikely to result in large degrees of polymerization. In addition, thepolymerization rate is proportional to the square root of the initiatorconcentration. As a result of these two considerations, we opted tosynthesize macromolecules that contained a high ratio of photoinitiatorsubstituents to recognition substituents.

A standard curve of initiator absorbance as a function of concentrationwas prepared. The solvent was MES buffer at pH 6 (0.1 M MES, 0.5 MNaCl). Absorbance measurements of purified macroinitiator solutions (100μg/ml in MES buffer) were made, and the standard curve was used todetermine the concentration of the initiator in the unknown productsolution. The measured absorbance value was 0.7 for 0.5 ml of 100 μg/mlof the macroinitiator.

HABA assays (Green et al.) were used to determine the average number ofNeutravidin substituents and verify retention of biotin-bindingcapability. HABA forms a complex with avidin that absorbs at 500 nm.Biotin displaces HABA due to the stronger affinity of the biotin-avidininteraction and as a result the absorbance at 500 nm decreases. Theamount of biotin that had to be added to a solution of the purifiedmacroinitiator (in MES buffer at pH 6) in order to make the shoulder inthe absorbance spectrum disappear allowed calculation of the amount ofavidin that was covalently attached to the macroinitiator.

¹H NMR spectra were collected using samples comprised of 20 mg of themacroinitiator dissolved in 0.7 ml of deuterium oxide. The water peakwas pre-saturated for collection of the spectra. NMR analysis requires alarger scale synthesis (60-fold compared with the description in theMethods section), and a methanol precipitation step was more practicalin this case than purification with molecular weight cutoff (MWCO)filters. MWCO filters performed well for solutions of this product withconcentrations of 1 mg/ml or less, but they did not provide goodseparation with higher product concentrations.

Arrays. Optical thin film biosensor (Inverness Medical-Biostar) surfaceswere prepared and oligonucleotides were covalently coupled to thesurfaces through hydrazone linkages according to previously publishedprocedures. (Zhong, X. et al., Single-nucleotide polymorphism genotypingon optical thin-film biosensor chips. Proc. Natl. Acad. Sci. U.S.A. 100,11559-11564 (2003)). In this reference, wafers were spin-coated with alayer of T structure aminoalkylpolydimethylsiloxane (TSPS) and poly(Phe-Lys) was passively adsorbed to the TSPS layer to facilitatecovalent attachment of biomolecules.

To make the dilution chip shown in FIG. 6 a, spotting solutions weredelivered to the surface in 60 mL droplets using a robotic microarrayer(custom instrument, Biodot); the resulting spots measured approximately600 μm in diameter. Biotinylated oligos were diluted into spottingsolutions containing unlabeled oligos so that the total oligoconcentration remained constant across the array.

Enzymatic amplification. Detection of biotinylated oligos usingenzymatic methods was accomplished by incubating thin film biosensorswith 50 μl of a 1 mg/ml solution of anti-biotin-HRP conjugate (JacksonImmunoResearch Laboratories) in a buffer comprised of 5×SSC, 0.1% SDS,and 0.5% BlockAid™ (Invitrogen-Molecular Probes) for 10 minutes.Following thorough rinsing with 0.1×SSC buffer, thin film biosensorswere incubated with 60 μl of a TMB/dextran solution (BioFX Laboratories)for 15 minutes. After rinsing with distilled water, thin film biosensorswere visually inspected for a color change from gold to blue (Jenison,R., Yang, S., Haeberli, A., Polisky, B. Interference-based detection ofnucleic acid targets on optically coated silicon. Nature Biotechnol. 19,62-65 (2001).

Polymerization-based amplification. Detection of biotinylated oligosusing photopolymerization was accomplished by incubating thin filmbiosensors with 50 μl of a 1.6 mg/ml solution of a macrophotoinitiatorin a buffer comprised of 5×SSC, 0.1% SDS, and 0.5% BlockAid™(Invitrogen-Molecular Probes) for 10 minutes. No steps were taken toprotect the macroinitiator from ambient light; it is stable underambient light conditions. Immediately following rinsing with 0.1×SSCbuffer, while the surfaces were still wet, 50 μl of an argon-purgedmonomer solution (97% by weight hydroxyethyl acrylate and 3% by weightethyleneglycol dimethacrylate crosslinker, each triply distilled toremove inhibitors) was pipetted over the entire array. Polymerizationfrom spots containing biotin and bound macrophotoinitiator wasaccomplished using a 10 minute dose of 5 mW/cm² UV light centered around365 nm from a Blak-Ray B Series-100A lamp. The percent of thecrosslinker in the monomer formulation and the dose of radiation wereoptimized by systematic variation of each. Polymerized arrays werephotographed with a digital camera without any rinsing or otherpost-polymerization treatment. The resulting hydrogel was susceptible todamage from rinsing to remove unreacted monomer

Further details are given in Sikes, H. S.; Hansen, R. R.; Johnson, L.M.; Jension, R.; Birks, J. W.; Rowlen, K. L.; Bowman, C. N. NatureMaterials 2007, 7, 52-56, hereby incorporated by reference.

Example 12 Recognition Between Biotinylated Oligonucleotide Arrays andStreptavidin Functionalized with Photoinitiator

A rapid (20 minute), non-enzymatic method of signal amplificationutilizing surface-initiated photopolymerization is presented in glassmicroarray format. Visible light photoinitiators covalently coupled tostreptavidin were used to bind biotin labeled capture sequences.Amplification was achieved through subsequent contact with monomersolution and the appropriate light exposure to generate 20 to 240 nmthick hydrogel layers exclusively from spots containing the biotinlabeled DNA. An amplification factor of 10⁶-10⁷ was observed as well asa detectable response generated from as low as ˜10⁴ labeledoligonucleotides using minimal instrumentation, such as an opticalmicroscope or CCD camera. This corresponds to a visual limit of about 10biomolecules/μm².

Polymerization-based amplification methods were extended to visualdetection of biotin-labeled DNA functionalized on glass microarraysurfaces with the use of photoreducible dyes which initiate upon visiblelight exposure. We chose Eosin Isothiocyanate (EITC) as ourphotoinitiator due to its favorable absorption characteristics anddemonstrate its ability to transduce biomolecular recognition into amacroscopically observable response in a highly sensitive manner. Atertiary amine coinitiator is added to bulk monomer to generate freeradicals for polymerization of a polyethylene glycol diacrylate (PEGDA)monomer solution. Similar systems have previously been reported forsurface initiated polymerization from silica nanoparticles (Satoh, M.;Shirai, K.; Saitoh, H.; Yamauchi, T.; Tsubokawa, N. Journal of PolymerScience: Part A: Polymer Chemistry 2005, 43, 600-606), fromaminosilinated glass surfaces for photopatterning applications (Kizilel,S.; Perez-Luna, V. H.; Teymour, F. Langmuir 2004, 20, 8652-8658;Kizilel, S.; Sawardecker, E.; Teymour F.; Perez-Luna, V. H. Biomaterials2006, 27, 1209-1215; Kizilel, S.; Perez-Luna, V. H.; Teymuor F.Macromolecular Theory and Simulations 2006, 15, 686-700), and in cellencapsulation studies (Cruise, G. M.; Hegre, O. D.; Scharp, D. S.;Hubbell, J. A. Biotechnology and Bioengineering 1998, 57, 655-665).Demonstration of DNA detection from polymerization-based signalamplification on general microarray surfaces enables use as a detectionmethod in applications commonly found in microarray technology, such assingle nucleotide polymorphism screening (Urakawa, H.; Noble, P. A.; ElFantroussi, S.; Kelly, J. J.; Stahl D. A. Applied and EnvironmentalMicrobiology 2002, 68, 235-244; Peterson, A. W.; Wolf, L. K.;Georgiadis, R. M. Journal of the American Chemical Society 2002, 124,14601-14607). and should be directly applicable to implementation oncommercially available microarray platforms (Hardiman, G.Pharmacogenomics 2004, 5, 487-502).

Microarray Fabrication. Commercially available amino or aldehydefunctionalized glass substrates were purchased from CEL associates andall slides were stored in vacuum at room temperature. The surface wasspotted through pin deposition using a solid pin to deposit ˜570 μmdiameter spots and a quill pin for ˜100 μm diameter spots at 75%humidity. An oligonucleotide sequence (5′amino-CATCACACAACATCACACAACATCACGTATATAAAACGGMCGTCGAAGG-3′ TEG biotin)(Operon) was spotted at an overall concentration of 20 μM on aldehydesubstrates in the spotting buffer (3×SSC, 0.05% SDS) using a VersArrayChipWriter Pro system made by Bio-Rad Laboratories. The identical,unlabeled capture sequence was spotted on the surface at a concentrationof 4 μM, which has been shown to be optimal for hybridizations. Theseslides were left in humidity for 24 hours. Aminosilane slides werespotted with 4 μM concentrations of 5′ biotin, 5′ Cy3, or unlabeledversions of this same sequence with varied concentrations of labeledsequences present for fabrication of dilution chips. These were left inhumidity for 30 minutes, then dried in an oven at 80° C. for 2 hours andfinally crosslinked to the surface using a 254 nm light. All spottedslides were stored at −18° C. until use.

Photoinitiator Synthesis. The visible light photoinitiatorEosin-5-Isothiocyanate (Invitrogen) was functionalized directly ontoexternal lysine residues of streptavidin through formation of a thioureabond (Hermanson, G. T. Bioconjugate Techniques; Academic Press: SanDiego, 1996) according to the reaction shown in scheme 10:

Streptavidin was dissolved in carbonate buffer (0.10 M NaCO₃, pH 9) at aconcentration of 10 mg/mL. A 10 mg/mL solution of EITC in DMSO wasprepared and immediately added to streptavidin at a volumetric ratio of1:10. The solution was reacted for 8 hours at 4° C., then diluted to astreptavidin concentration of 1 mg/mL in 1×PBS and purified using gelfiltration. The product was characterized with conventional UV-Visspectroscopy, and the characteristic peak from EITC at 525 nm wascompared to the characteristic protein peak at 280 nm according toequation 3:

$\begin{matrix}{{n_{EITC}/n_{SA}} = \frac{{Abs}_{{EITC},525}/ɛ_{{EITC},525}}{\left( {{Abs}_{{SA},280} - {Abs}_{{EITC},280}} \right)/ɛ_{{SA},280}}} & (3)\end{matrix}$

Measuring 0.2 mg/mL solution of the product diluted in 1×PBS buffer, anaverage photoinitiator to protein ratio of 2.3 was observed. The productwas stored at 4° C. and protected from light exposure until further use.

Microarray Functionalization of Photoinitiator Product. Spotted slideswere blocked with 2 weight percent dry milk in ddH₂0 for 2 hours toprevent nonspecific adsorption of the photoinitiator product to thesurface. Slides were then rinsed with water and contacted with 200 μL at1 μg/mL of visible photoinitiator product in 1×PBS and 5× Denhartssolution for 30 minutes. Slides functionalized with streptavidin-EITCwere either placed in boiling water for two minutes or washed in TNTsolution (1M NaCl, 0.1M Tris, 0.1 wt % Tween 20) to remove nonspecificprotein adsorbed on the surface. Slides were rinsed in ddH₂O and allowedto dry.

Surface-Initiated Photopolymerization. MEHQ inhibitor was removed frompoly (ethylene glycol) diacrylate (PEGDA) (M_(n)˜575 Da) with the use ofa dehibit column (Sigma). Methyl diethanol amine (MDEA) and1-vinyl-2-pyrrolidinone were used as received. The reactive monomersolution consisted of 25 wt % PEG(575)DA, 225 mM MDEA, 37 mM1-vinyl-pyrrolidinone in a ddH₂O solvent. The pH was about 9. 300 μL ofone of the monomer solutions was purged with argon, then contacted withthe surface using a Whatman Chip Clip. Slides were placed in an argonchamber that was continuously purged for 5 minutes prior to radiation. A400-500 nm Novacure collimated light source was used to radiate thestreptavidin-EITC functionalized slides at 8 mW/cm² for 20 minutes. Thesamples were gently rinsed with ddH₂O to remove unreacted monomer andthen dried with argon.

Post-Polymerization Surface Characterization of Microarray Surface. Asurface profilometer (Dektak 6M) was used to obtain height profiles ofthe films that were visible on the array after drying the chips suchthat the hydrogels was completely dehydrated. Stylus force was set at aminimum (1 mg) to minimize mechanical deformation of the hydrogel layersfrom the 12.5 μm diamond stylus tip. An infrared microscope was used toobtain IR spectra of surface bound moieties. An Agilent Technologies DNAMicroarray Scanner was calibrated at specific settings (100% PMT Gain)with a Cy3 calibration chip obtained from Full Moon Biosystems and usedfor fluorescent imaging.

Fluorescent Imaging of Photoinitiators on Surface. As an initialcharacterization of the amplification processes, aldehyde functionalizedmicroarray surfaces were spotted with rows of 5′ amino, 3′ biotinfunctionalized capture sequences and rows of 5′ amine capture sequenceswithout the biotin label. Upon incubation of 1 μg/mL concentrations ofStreptavidin-EITC, photoinitator molecules should be localized to thelabeled capture sequences with the strong affinity ofbiotin-streptavidin complex (K_(d)=10⁻¹⁵). Eosin, a weak fluorophorewith a fluorescence quantum yield of 0.19 (Weber, G.; Teale, F. W. J.;Transactions of the Faraday Society 1957, 646-655), has a strongabsorbance at 532 nm, an excitation source used by most conventionalmicroarray scanners. Thus, fluorescent scanning was used to quantify thenumber of biotin-streptavidin binding events on the surface as well asbackground from non-specific protein interactions. A fluorescent imageof a microarray containing 3′ biotin labeled capture sequences andunlabeled capture sequences after streptavidin-EITC incubation but priorto amplification shows that the average fluorescent signal of positivespots containing streptavidin-EITC molecules is 26,000 while unlabeledsequences show low fluorescent counts of 400 and average background of350 fluorescent counts, close to machine noise (325). Fluorescentintensities show uniform signal both within individual spots (StandardDeviation=3260) and between duplicate spots (Standard Deviation=3060).

Upon quantification of the fluorescent intensity at the positive spotsminus background absorbance, there are 480+/−60 eosin fluorophores perμm². Because the number of fluorescent eosin molecules per streptavidinprotein was determined as 2.3, there is an average of 200biotin-streptavidin binding events per μm² at positive sites on thebiochip. The variance in signal intensity of <15% relative standarddeviation both in average fluorescent counts between positive spots andbetween counts within individual spots, giving evidence of relativelymonodisperse allocation of photoinitators on the surface. Thismonodispersity is achieved primarily from use of optimized spottingbuffer during the printing process and contacting the streptavidin-EITCsolution on the surface without the use of a coverslip. Because of themonodispersity attained here, a uniform number of radicals is eventuallygenerated across the positive spots during the surface initiatedphotopolymerization process to aid in generating polymer films ofuniform thickness.

Surface Initiated Photopolymerization with Visible Light. Uponfunctionalization of streptavidin-EITC, rapid amplification wasperformed by contact with monomer and light exposure to generatepropagating radicals for amplification at the solid-liquid interface.Using a 25 wt % solution of PEG(575)DA monomer with a 225 mM MDEAcoinitiator and 37 mM 1-vinyl-2-pyrollidone accelerator in an aqueoussolvent has been shown to form stable, micron thick hydrogels on EITCfunctionalized aminosilinated glass surfaces (Kizilel, S.; Perez-Luna,V. H.; Teymour, F. Langmuir 2004, 20, 8652-8658). The presence ofdifunctional acrylate yields pendant double bonds in propagating polymerchains that may crosslink with other propagating chains, thussuppressing chain termination rates and causing large amounts of highmolecular weight polymer to be generated at positive sites.

Attachment of the PEG hydrogel to the surface is achieved throughtermination reactions between surface stabilized eosin radicals and bulkradicals present on PEGDA chains (Kizilel, S.; Perez-Luna, V. H.;Teymour, F. Langmuir 2004, 20, 8652-8658). In this application, strongattachment of the polymerized hydrogel layer to the surface was criticalto avoid delamination from swelling during washing to remove unreactedmonomer. After amplification using visible light (400-500 nm, 8 mW/cm²,20 minutes) and subsequent washing, highly visible, surface stabilizedhydrogel films have been observed exclusively over biotinylated sites.The stabilization to the surface is achieved here through the highaffinity streptavidin-EITC binding to covalently bound biotin. To verifythat the macroscopic response observed was due to surface initiatedphotopolymerization of the hydrogel precursor solution at biorecognitionsites, the visible polymer spots on the microarray surface werecharacterized through FTIR analysis. Film deposition of sub micron-scalethicknesses yields an IR spectra characteristic of the polymeric moietystabilized to the glass surface above a wavenumber of 2000 cm⁻¹. Anoptical microscope image of spots on the array shows surface initiatedpolymerization of a PEG hydrogel networks exclusively at biorecognitionsites. The microscope image shows the consistent formation ofwell-defined polymer spots only where 3′ biotin labeled capturesequences were present. Non-specific polymerization from the backgroundor from unlabeled capture sequences is not observed. IR spectragenerated from positive spots on the chip and at regions outside thespots also gives evidence of the surface initiated polymerization of aPEG hydrogel networks exclusively at biorecognition sites. Upon theformation of a polymer layer over biotinylated oligonucleotide spots,infrared peaks characteristic of hydroxy groups (broad peak from 3200 to3550 cm⁻¹) and alkyl groups (peak at 2900 cm⁻¹) present in PEG hydrogelbecome clearly visible. In areas on the chip outside the visible spots,no peaks are detectable.

To verify that these films were indeed polymerized only due tobiotin/photoinitator binding, monomer contact, and light, severalnegative controls were performed on glass surfaces. No polymerizationwas observed without incubation of the initiator or without lightexposure, consistent with what was previously observed on Biostar chips(Sikes, H. S.; Hansen, R. R.; Johnson, L. M.; Jension, R.; Birks, J. W.;Rowlen, K. L.; Bowman, C. N. Nature Materials 2007,doi:10.1038/nmat2042).

Film Thickness Measurement and Amplification Factor Determination. Tocharacterize the polymer films further on the biochip surface, filmthicknesses were measured through the use of profilometery. Such ananalysis will allow for investigation of the uniformity of the polymerfilm thickness both between positive spots and within each spot, as wellas an estimation of the number of monomers polymerized at each positivelocation. With the quantification of both the number of binding eventsof streptavidin-EITC on the surface and the film thickness of theresulting polymer films, an amplification factor, defined as the averagenumber of propagation reactions occurring per biorecognition event, isreadily estimated. FIGS. 7 a and 7 b show profilometry scan across spotscontaining 3′biotin labeled capture sequences (A) and unlabeled capturesequences (B). Both rows contained capture sequences at a surfacedensity of 10² capture sequences/μm². Average film thickness generatedfrom the 3′biotin labeled capture sequence was 140 nm, with a standarddeviation of 20 nm between spots and a similar standard deviation of 20nm between film thickness measurements within a single spot. Nonspecificamplification has been eliminated in the amplification system both inthe background and from unlabeled DNA. No signal was detectable aboveprofilometer error (10 nm) from spots containing the unlabeled capturesequences on the same biochip. FIG. 7 a shows a representativeprofilometry scan across a section of the biochip surface containingpositive spots signaling the presence of 3′ biotinylated capturesequences. Apparent in FIG. 7 a are well-defined step functionsgenerated from the polymer films, in 570 μm spots ranging from 110 nm to160 nm in thickness. No significant change of spot diameter from thepolymerization was incurred, as determined from comparison of spotdiameters from the fluorescent image before amplification with theprofilometry measurement after amplification. The uniformity measured ineach peak was due to monodispersity of photoinitiator allocationobserved from the fluorescence measurements. However, the 14% relativestandard deviation between peaks measured in FIG. 7 a may be due to thesmall variation in photoinitator surface density, small variations inlight intensity across the surface, impurities in the monomer solution,damage to the polymer film during washing, or from the highly amplifiednature of free radical polymerizations.

Despite the noted variation in film thickness, the well-defined peakheights measured in FIG. 7 a still allow for an estimation of the numberof monomers polymerized in each spot. Film thicknesses averaging 0.14 μmthick in a 570 μm diameter spot give a hydrogel volume of 3.67×10⁴ μm³.Taking the density of poly(ethylene glycol) (1.2 g/cm³) and the averagemolecular weight of each repeat unit (575 g/mol), 4.61×10¹³ monomerswere polymerized per spot (2×10⁸ monomers/μm²). As determined throughfluorescence, streptavidin-EITC binds 200 biotins/μm². Dividing thenumber of monomers per μm² by the number of biorecognition events perμm² gives a 1×10⁶ amplification observed on this surface. Thisamplification factor is comparable to 40 cycles of PCR amplification(Parsons, G. J. Clin. Immunoassay 1988, 11, 152-158).

Dilution Chips for Sensitivity Analysis. To investigate sensitivity ofthe visible light amplification system, a biochip containing a largevariation of labeled target oligo surface densities was developed onaminosilated glass surfaces. Aminosiliated surfaces were chosen infabrication of dilution chips as opposed to aldehyde modified glassbecause a larger range of oligonucleotide surface densities wereobtainable on aminosilated surfaces. On these dilution chips, thesurface concentration of labeled oligo decreased with each row of spots.To achieve this dilution, a constant overall spotting concentration ofoligonucleotide at 4 μM was desired with the amount of labeled oligodecreasing from 4 μM to 1.25 nM. Using Cy3 as the label at thesespotting concentrations followed by fluorescent scanning with a 532 nmexcitation source, surface densities were determined to range from 10⁴markers/μm² to <5 markers/μm². The most dilute rows of labeledoligonucleotide were assumed to be 5 markers/μm², the quantifiabledetection limit on the scanner. In actuality, the surface density islower. The final row represents unlabeled oligonucleotide only, as theoligo on the surface shows a small amount of background fluorescencemaking these spots visible under a scanner. Using a quill pin forspotting, spots are consistently 100 μm in diameter, thus the equivalentnumber of capture oligos in a spot on the surface ranges between 0.2femtomoles to ˜60 zeptomoles in the most dilute spots.

In fabrication of the biotin dilution chips, the end label was switchedfrom Cy3 to biotin, and assumed that nearly identical surface densitieswould be obtained with the respective spotting concentrations. Becausecovalent attachment of photoinitiator to the surface has been shown tobe required for stable hydrogel layers to remain on the surface duringswelling. (Kizilel, S.; Perez-Luna, V. H.; Teymour, F. Langmuir 2004,20, 8652-8658; Revzin, A.; Russel, R. J.; Yadavalli, V. K.; Koh, W. G.;Deister, C.; Hile, D. D.; Mellott, M. B.; Pishko, M. V. Langmuir 2001,17, 5440-5447) 254 nm light exposure was used to form covalentattachment of biotinylated oligonucleotide to the aminosilane layerusing a 254 nm light source at an exposure which was known to crosslinkthe oligo to the surface. A fluorescent image of the dilution chip uponincubation of the streptavidin-EITC visible light initiator at 1 μg/mLwas obtained. A fluorescent signal gradient is observed, giving evidenceof allocation of decreasing amounts of photoinitiator at oligo spotscorresponding with decreasing label density. Incubation at 1 μg/mL ofstreptavidin-EITC was considered sufficient for amplification,incubating with higher concentrations may lead to non-specificadsorption of initiator to the surface, either limiting the sensitivityof the amplification or giving the potential to amplify background aswell the positive spots. A fluorescent signal that is significantlydifferent than the negative column (unlabeled oligo) can be seen down tothe 12^(th) column, corresponding to ˜0.1 attomoles of surface boundbiotinylated oligo. The last three columns containing biotinylatedoligonucleotide, as well as the final negative column all show asimilarly small fluorescence level due to non-specificprotein-oligonucleotide electrostatic interaction, thus non-specificprotein adsorption has been minimized but not completely eliminated inthis system.

Detection Limit Determination Using Polymerization Based Amplification.The same hydrogel precursor solution as previously described was appliedto the microarray dilution chips with a 400-500 nm, 8 mW/cm² lightexposure. Light intensity and exposure time for use on the dilution chipwere chosen from FTIR bulk polymerization studies that showed completedouble bond conversion of this hydrogel solution as observed at 6120cm⁻¹ with streptavidin-EITC concentrations ranging from 10⁻⁵ M to as lowas 10⁻⁶ M. These concentrations represent the local concentrations ofEITC at different rows of positive spots on the dilution chips, giventhe photoinitiator surface density as determined through fluorescenceand assuming a 10 nm film over each spot. After 20 minutes from thebeginning of the polymerization, final conversion was reached with a10⁻⁶ M solution of streptavidin-EITC, representing spots on the dilutionchip with the smallest amounts of initiators present. Thus, with thisexposure it can be assumed that the final conversion would be achievedwhen initiating polymerization from all rows at the surface of ourbiochip.

After polymerization on the dilution chips and washing, spots becameconsistently visible under an optical microscope or CCD camera to as lowas ˜0.4 attomole levels of biontylated oligo (10 labeled oligos perμm²), a macroscopically observable response generated from biotinlabeled oligonucleotides at surface densities approaching thequantifiable detection limits of most microarray scanners.

TABLE 2 Row 1 2 3 4 5 6 7 8 9 10 11* 12 13 14 15 16 Moles of 237 234 222207 184 121 109 59 1.4 0.68 0.30 0.17 0.08 0.08 0.08 0.00 5′Biotin- DNA(10⁻¹⁸) +/− Error 10 7 5 10 10 7 4 4 0.04 0.03 0.02 0.00 0.00 0.00 0.000.00 Film 0.20 0.13 0.20 0.20 0.24 0.20 0.22 0.24 0.11 0.06 0.08 0.080.01 0.02 0.02 0.02 Thickness (μm) +/− Error 0.09 0.05 0.07 0.08 0.110.08 0.08 0.08 0.06 0.03 0.04 0.05 0.01 0.01 0.02 0.01 *Visual DetectionLimit

This detection limit was repeatable using different batches ofstreptavidin-EITC, monomer, and dilution chips. Average film thicknessesgenerated from each row on dilution chips are presented in Table 2.Table 2 is a summary of film thicknesses generated from specificquantities of biotinylated oligonucleotide sequences on each of 16 rowson dilution chips using photopolymerization based amplification.

Data was averaged from different batches of reagents and materials thatwere fabricated several months apart. FIGS. 8 a and 8 b show filmthicknesses obtained for rows containing different amounts ofbiotinylated DNA on the dilution chip using the visible light initiationsystem (representative profilometry scan down the rows of the dilutionchip). For higher numbers of biotinylated oligonucleotide (˜0.2femtomoles to ˜60 attomoles), film thicknesses averaging from 0.2 to 0.3microns have been observed (FIG. 8 a). At lower concentrations ofbiotinylated DNA, film thicknesses became less then 0.1 microns thickbut still remained consistently visible under an optical microscope or aCCD camera down to the 11^(th) row, generated from ˜0.4 attomoles ofbiotinylated oligo (A), below which no other spots were visible. Usingprofilometry as a means of detection, a statistical difference of filmthickness from the negative control has been detected down to the12^(th) row, containing ˜0.1 attomoles (FIG. 8 b).

As is shown in FIGS. 8A and 8B a saturation region appears to be presentin regards to responses generated from 0.2 femtomoles to 60 attomoles.Peaks generated from 1 to ˜0.1 attomoles are consistently less thick andless visible. With profilometry, a peak generated from each of thesixteen rows on the dilution chip is detectable, including that from thenegative row containing unlabeled oligonucleotide. Because of theelectrostatic interaction between streptavidin-EITC and oligonucleotidethat was observed from fluorescence scanning, these peaks may be due toamplification from the non-specific allocation of initiator ontooligonucleotide spots. From FIGS. 8 a and b, a statistical difference infilm thickness from the negative control corresponding to a 99%confidence level can be shown from as low as 0.1 attomoles (the 12^(th)row). At these lower levels, the estimated amplification factorincreases to 10⁷. Peaks from samples below 0.1 attomoles show nosignificant difference in film thickness from the negative control; thiscorresponds to what was observed from fluorescence intensities thatreveal the same amounts of initiator allocation prior to amplificationon all spots containing less then 0.1 attomoles. Currently, thenon-specific interaction is the limiting factor in regards to thesensitivity of the system.

We have demonstrated a method of amplifying a biorecognition event onglass microarray surfaces at high sensitivity through functionalizingsurface bound oligonucleotides with photoinitiators, followed by contactof monomer solution and appropriate visible light exposure. Thehydrogels formed at biorecognition sites polymerized to sub-micron levelthicknesses in a matter of minutes, were well-defined with uniformthicknesses, and showed stable attachment to the glass surfaces uponwashing to remove unreacted monomer. Minimal background or non-specificamplification was observed on the surfaces. While this amplificationsystem was demonstrated for the detection of biotin-avidin binding, sucha system can be readily extended to the detection of complementarynucleic acid hybridization through the use of chemical or enzymaticlabeling approaches (Zhong X.; Reynolds, R.; Kidd, J. R.; Jenison, R.;Marlar, R. A.; Ward, D. C. Proc. Natl. Acad. Sci. 2003, 100,11559-11564). For example, polymerase enzymes may be used for labeling3′ ends of hybrid DNA on a microarray surface with labeled dNTPs in aprimer extension reaction (Pastinen, T.; Raitio, M.; Lindroos, K.;Tainola, P.; Peltonen, L. Syvanen A. C. Genome Research 2000, 10,1031-1042; Hultin, E.; Kaller, M.; Ahmadian, A.; Lundeberg, J. NucleicAcids Research 2005, 33, e48). Using biotinylated dNTPs in such areaction would then render this surface the same as the model surfacesstudied in this example. Polymerization-based amplification ofcomplementary DNA hybridization (1 μM target hybridization of K-Rasbiomarker) has been demonstrated using this approach.

The generation of macroscopically observable hydrogel spots due to thepresence of as low as sub-attomole amounts of labeled genetic materialon microarray substrates should allow for at least a sensitive,positive/negative determination to be made without the use of detectionequipment. With the rapid, inexpensive, and robust characteristics ofthis amplification method, photopolymerization for detection ofmolecular recognition holds considerable potential, as amplification ofat least 10⁶-10⁷ will help facilitate use of molecular diagnosticapplications in clinical settings.

Further details are given by Ryan R. Hansen, Hadley D. Sikes, andChristopher N. Bowman, Biomacromolecules, 2008, 9(1) pp 355-362; whichis hereby incorporated by reference.

Example 13 Quantification of Oligonucleotide Surface Concentrations andIncorporation of Fluorescent Nanoparticles into Polymer Films

Quantitative characteristics of a visible light polymerization-basedamplification system were investigated for use in biodetection assaysrequiring sensitive, sequence-specific detection of polynucleotides. Theapproach taken involves fabricating biochip surfaces witholigonucleotide spots containing controlled concentrations ofimmobilized, biotinylated DNA targets capable of capturing proportionalamounts of streptavidin-initiator, followed by contact with a monomersolution and a discrete light exposure. Determination of the amounts ofamplification generated at each target concentration is evaluated withfilm thickness measurements, allowing for calibration of film thicknesswith DNA concentration. While amplification is evaluated in asolid-state biosensor format, the results should also be significantwhen considering implementation of this amplification system tosolution-based assays as well.

To implement inexpensive detection instrumentation for quantification,it is desirable that PEG films with quantitative thicknesses generatedfrom polymerization-based amplification be correlated with a measurablesignal consistent with inexpensive methods of detection. Direct filmthickness measurements at the nanometer level may be infeasible inclinical settings due to the expense of the necessary instrumentation(profilometry, ellipsometry, atomic force microscopy). Developing thisamplification system towards generating an amplified fluorescent signalthat corresponds with film thickness is particularly desirable. This isdue to the wide range of instrumentation typically available forcharacterizing biomolecule interactions using fluorescent measurements.For microarrays, such instrumentation ranges from laser based microarrayscanners using photomultiplier tube (PMT) detectors (detection limit<0.05 fluors/μm², 10⁶ dynamic range), costing hundreds of thousands ofdollars to fluorescent readers employing inexpensive excitation sources(such as a 10 mW, 532 or 635 nm laser pointers) and CMOS detectors withassociated equipment cost less than $1000 and with minimal powerrequirements (detection limit ˜5000 Cy3 fluors/μm²). With theseconsiderations in mind, an initial demonstration of usingpolymerization-based amplification to achieve an amplified, quantitativefluorescent signal is demonstrated herein.

One approach to coupling fluorescent signal with polymer growth is toinclude the fluorescent moiety in the monomer formulation such that itis copolymerized or encapsulated into the crosslinked polymer network.With the commercial availability of several types ofacrylated-fluorophores, copolymerization of molecules such asfluorescien-o-acrylate into PEG-based polymer films on a solid substratehas been demonstrated as a method of coupling a fluorescent response tohydrogel formation (Kizilel, S.; Sawardecker, E.; Teymour F.;Perez-Luna, V. H. Biomaterials, 2006, 27, 1209-1215). In application tophotopolymerization-based amplification, several limitations to suchapproaches are inherent, namely non-specific initiation of bulk monomerdue to photoreduction reactions occurring between a photoexcitedfluorophores and amine coinitiators; secondly, decreased initiationrates from the surface-bound eosin initiators due to light attenuationto the surface caused by the absorbing fluorophore; and finally,saturation of fluorescent signal after amplification due to quenching offluorescent molecules at higher concentrations. An alternative tofluorescent acrylates are commercially available, 20 nm diameterpolystyrene microspheres that encapsulate ˜10² hydrophobic fluorophoresper particle (Invitrogen). These fluorescent particles mitigate many ofthe above limitations largely due to the fact that they eliminatefluorophore contact with their surrounding environment and remainstrongly fluorescent.

Dilution chip development. Oligonucleotide arrays were characterized byspotting decreasing concentrations of 5′Cy3 functionalized 50 baseoligonucleotides (Operon) on aminosilated glass (CEL Associates) using a375 μm diameter stealth solid pin (Arraylt) and quantifying the surfaceconcentrations through fluorescence scanning (Hansen, R. R.; Sikes, H.D.; Bowman, C. N. Biomacromolecules, 2008, 9, 355-362). The dilutionarray contained decreasing amounts of 3′ biotinylated oligonucleotidediluted in a solution of unlabeled oligonucleotide capture probes suchthat the overall concentration of oligonucleotide remains constant at 4μM. Dilution chips were fabricated to capture a three order of magnituderange of surface densities with an even distribution of intermediatesurface densities to allow for a comprehensive evaluation of filmthicknesses generated from polymerization based amplification. Thespecific concentrations used were: 4 μM, 3 μM, 2 μM, 1 μM, 530 nM, 270nM, 130 NM, 78 nM, 53 nM, 40 nM, 30 nM, 20 nM, 10 nM, 5 nM, 2.5 nM, 0nM.

In actual fabrication, 5′ biotin triethylene glycol was replaced as thelabel. Slides were dried at 85° C. for 1 hour and then exposed to 254 nmlight to couple capture sequences onto the amine attachment layer.Slides were then washed with water to remove buffer salts and stored at−20° C. until further use.

Functionalization and characterization of streptavidin-eosinisothiocyanate. Streptavidin-eosin isothiocyanate (SA-EITC) wassynthesized, purified, and characterized according to previouslypublished protocol (Hansen et al., 2008). Dilution chips werefunctionalized with SA-EITC by applying a mixture of unlabeledstreptavidin (Pierce) and SA-EITC at 1 μg/mL for concentration for 30minutes followed by washing with TNT solution (1M NaCl, 0.1M Tris, 0.1wt % Tween 20) and then drying with N₂. Preamplified, SA-EITCfunctionalized dilution chips were characterized using an AgilentMicroarray scanner (green channel, 100% PMT) calibrated using a Cy3/Cy5calibration chip (Full Moon Biosystems). Denharts solution was used as ablocking agent.

Monitoring of bulk polymerization kinetics. The initiating capability ofSA-EITC in various monomer formulations was verified by monitoring bulkpolymerization through the use of Fourier transform infrared (FTIR)spectroscopy. The double bond conversion at 6120 to 6210 cm⁻¹ wasmonitored using monomer formulations (25 wt % PEGDA, M_(n)=575 Da, 225mM methyl diethanol amine coinitiator, 37 mM 1-vinyl-2-pyrrolidinone, inH₂O, pH 9) containing 50 ng/mL SA-EITC initiator (1 μM eosinconcentration) and specified concentrations of Nile Red FluoSpheres(Invitrogen) in a sample with a 2 mm pathlength.

Polymerization based amplification. Monomer solution (25 wt % PEGDA,M_(n)=575 Da, 225 mM methyl diethanol amine coinitiator, 37 mM1-vinyl-2-pyrolldinone, in H₂O, pH 9) was purged with argon and then 500μL was contacted with the dilution chip surface using a Whatman ChipClip. Collimated, 495-650 nm polychromatic light was used from anActicure light source at an intensity of 10 mW/cm² to initiatepolymerization. This was achieved with the use of an in-house 350-650 nminternal interference filter and a 495 nm longpass filter (EdmondOptics) applied to the end of a collimating lens attached to the end ofa light guide. Argon was purged from the atmosphere for 5 minutesfollowed by exposure of light for the desired time period. Afteramplification, unreacted monomer was removed from the surface byremoving the dilution chip out of the chip clip and then gently washingthe surface with water.

Polystyerene microspheres encapsulating fluorescent molecules wereincluded in monomer solution for subsequent encapsulation into thehydrogel matrix during polymerization. 0.02 μm diameter,carboxy-modified Nile Red FluoSpheres (Invitrogen) were obtained at astock concentration of 4.5×10¹⁵ microspheres/mL containing 0.1% sodiumazide preservative. The azide preservative was removed with the use of100,000 Da dialysis columns (Spectra-Por), according to themanufacturer's protocol. The purified FluoSpheres were stored at 4° C.until further use. A 1:50 or 1:10 dilution of the stock solution wasmade into the established monomer solution. Light intensity wasincreased accordingly to account for light attenuation occurring fromthe FluoSpheres over the 1 mm thick monomer layer. The amplificationthen followed the protocol previously detailed.

Post-amplification characterization of dilution chips. Film thicknesseswere measured with a Dektak 6M profilometer using a 12.5 μm diamondstylus tip at a minimal stylus force of 1 mg to minimize polymer damagefrom contact with the pin. Brightfield and fluorescent pictures ofamplified dilution chips were obtained through the use of a Leicastereomicroscope calibrated with a Cy3/Cy5 calibration chip (Full MoonBiosystems). Nile Red fluosphere-functionalized polymer films on thesurface were further quantified with the use of an Agilent TechnologiesMicroarray scanner (Green channel, PMT 1%) Because of thewell-controlled photoinitiator to streptavidin ratio (3:1) achieved inthe streptavidin-eosin isothiocyanate (SA-EITC) synthesis (Hansen et al,2008), the visible light amplification system was chosen as the systemto use for quantifiable detection. Arrayed dilution chips containingdecreasing surface concentrations of 3′ biotin labeled oligos were usedto determine the amount of amplification achieved as a function oftarget oligo surface concentration, determined either through directmeasurement of film thickness or through measuring an amplifiedfluorescent signal. This approach of directly printing the biotinylatedtarget onto the surface serves as a model system (Wilcheck, M.; Bayer,E. A.; Livnah, O. Immun. Letters, 2006, 103, 27-32) to evaluate thecharacteristics of polymerization-based amplification. Such a system ismeant to represent a biochip after complementary duplex formation andlabeling that may be done either on chip (Pastinen, T. et al. Gen. Res.,2000, 10, 1031-1042; Hultin, E., Kaller, M.; Ahmadian, A.; Lundeberg, J.Nucleic Acids Res., 2005, 33, e48; Erdogan, F. et al. Nucleic AcidsRes., 2001, 29, e36) or in solution (Do. J. H.; Choi, D. K. Eng. LifSci. 2007, 7, 26-34). Thus, the surface density of the biotin labelshould be directly related to the target concentration, dependent on thehybridization efficiency between a target and its surface-boundcomplementary capture probe (typically, K_(hyb)=10⁹ M⁻¹) (Michel, W.;Mai, T.; Naiser, T.; Ott, A. Biophysical Journal, 2007, 92, 999-1004),and the labeling efficiency.

Dynamic amplification and tunable sensitivity. Upon functionalization ofa dilution chip with SA-EITC, dilution chips are imaged with afluorescent scanner to verify the allocation of initiators on thesurface at densities proportional to the specific biotin label densitiesat each site. Long exposure times of low intensity light were used tocompletely react monomer even from the spots containing the lowestinitiator concentrations. At long exposure times, long-wave visiblelight is desired to minimize or eliminate any nonspecific amplificationthat is characteristically observed from light sources with emissions inthe UV region. Thus, polymerizations using polychromatic green lightfrom a mercury arc lamp at intensities of 1 mW/cm² were performed. Thelow intensity light allows for slower degradation of initiator intoreactive free radicals at the surface, which is desired to achievedecreased termination rates. By examining several longer exposure timesin the amplification, times greater than 30 minutes were determined tobe required for obtaining maximum sensitivity.

Upon amplification of this system at 30 minutes, polymer films generatedfrom as low as 10 biotin markers/μm² become visually observable under anoptical microscope or to the unaided eye. Analysis of film thicknessesat each site showed that a corresponding dynamic profile was achievablefrom the range of 50 to 4800 biotin markers/μm². This was followed by asaturated region from 6,000 to 15,000 biotin markers/μm² with filmthicknesses all in the 230+/−20 nm region, independent of markerdensity, as shown in FIG. 9. The dynamic region of amplification,approaching two orders of magnitude, fits a logarithmic correlationwithin error, typically less than or equal to 20%. Further demonstratedin FIG. 9 is a tradeoff between assay time and sensitivity for thissystem. Exposing at times allowing for incomplete conversion of monomerinto polymer has the capability to shift the sensitivity of the system,as 5 minutes of exposure shifts sensitivity back to 2,000 biotinmarkers/μm². Such tunability allows the user capability to implementoptimal assay conditions depending on the required sensitivity of theapplication.

The increase in film thicknesses observed with higher biotin targetconcentrations is directly due to the corresponding increase ofphotoinitiator surface concentrations on binding with SA-EITC. Withhigher photoinitiator concentrations on the surface, an increased numberof propagating radical chains are generated during the polymerizationprocess. Such free radicals are capable of diffusing from the surfaceand through the forming polymer film thereby increasing the conversionof monomer into surface-bound polymer and ultimately increasing filmthicknesses. This trend is consistent with both modeling andexperimental findings of similar systems in the literature (Kizilel, S.;Perez-Luna, V. H.; Teymour, F. Macromol. Theory Simul. 2006, 15,686-700). After the polymerization reaches high conversions, radicaldiffusivity throughout the hydrogel membrane decreases and eventuallylimits the extent of the polymerization, causing saturation in filmthicknesses observed at spots containing higher concentrations oftargets. A secondary explanation of saturation may be due to highertermination rates associated with the higher initiator concentrations,which generate higher concentrations of reactive radicals, allowing forincreased termination by combination.

The dynamic dependence of film thicknesses with photoinitiator surfaceconcentrations allows for an additional method of controlling thesensitivity of this assay and also enables the ability to tune dynamicamplification to concentration regions of interest. By varying thebinding parameters on the surface, allocation of different amounts ofphotoinitiator to sites containing a constant amount of target isachievable. This potentially allows the user the ability to detect andquantify biotinylated DNA concentrations that may be either undetectablewith less efficient binding or in a saturated region with more efficientbinding.

A simple demonstration of this technique can be accomplished through theuse of a competitive binding technique involving incubating dilutionchips with solutions containing various ratios of unmodifiedstreptavidin (SA) with SA-EITC. Due to the binding of unmodified SA tobiotinylated DNA, the concentration of biotinylated DNA available forbinding with SA-EITC decreases with higher SA to SA-EITC ratios. Becauseeosin is a weak fluorophore, a decrease in fluorescent intensities atidentical biotin concentrations can be observed on dilution chipsfunctionalized with higher SA:SA-EITC ratios. This signals decreasedphotoinitiator concentrations at the same biotin concentrations, asshown in FIG. 10A. When incubating with higher SA:SA-EITC ratios, thefluorescent signal required for detection (˜2000) is achieved at spotscontaining higher biotin surface densities. Also, higher end spots withphotoinitiator concentrations corresponding to saturation fromamplification with out the addition of SA are shifted to quantifiableconcentrations at higher SA:SA-EITC ratios. The result of this is ashift in detection limits towards less sensitive detection, but also ashift in the concentration regions of dynamic amplification as shown inFIG. 10B.

In the case of detecting nucleic acid hybridization withpolymerization-based amplification, an alternative and perhaps morefeasible approach involves varying capture sequence densities. Capturesequence density has been shown both experimentally and through modelingconsiderations to be a crucial parameter for achieving optimal signalfrom hybridization (Halperin, A.; Buhot, A.; Zhulina, E. B. BiophysicalJournal, 2004, 86, 718-730; Pererson, A. W.; Heaton, R. J.; andGeorgiadis, R. M. Nucleic Acids Res., 2001, 29, 5163-5168). In effort toshift a saturated response generated from complementary target-capturehybridization to a quantifiable response, sites with less then optimalcapture probe densities could be printed. Such sites would then renderlower photoinitiator concentrations than optimal sites at the sametarget concentrations, potentially shifting a saturated response to aquantifiable response. With the high throughput capability of DNA chips,several sites of identical complementary capture sequences could beprinted at optimal conditions and successively less than optimalconditions within a single array, allowing for a quantifiable responseto be achieved over many orders of magnitude. The capability ofquantification at higher ends of the dilution chip extends the totaldynamic region observed from polymerization-based amplification to 50 to18,000 biotins/μm², well over two orders of magnitude. Assuminghybridization constants typical of complementary duplex formation onmicroarray surfaces (K_(hyb)=10⁹ M⁻¹) (Michel, W.; Mai, T.; Naiser, T.;Ott, A. Biophysical Journal, 2007, 92, 999-1004) and that each targetsequence can be labeled with a single biotin marker, the regions ofquantification amplification correspond to analyte concentrations in thenM to pM range.

Coupling fluorescent signal with polymer growth. To achieve fluorescentsignal gains using polymerization-based amplification, 20 nm diameterNile Red (532/575) FluoSpheres (ε₅₃₂=2.3×10⁶ cm⁻¹ M⁻¹) were added tomonomer solution at various dilutions. FIG. 11 shows the absorbancespectra of the monomer solution containing 90 nM fluorescentnanoparticles compared to that of a 10 μg/mL solution of SA-EITC. Thereis a considerable overlap in absorbance between the monomer solution andthe initiator at wavelengths in the 500-650 nm region used to initiatethe polymerization reaction, thus adsorption of light from fluorescentmonomer effectively decreases initiation rates from SA-EITC at constantlight intensity. This effect is observed when monitoring bulkpolymerization kinetics under initiation conditions similar to thoseused during on-chip amplification. The monomer conversion with time isshown in FIG. 12 using 2 μM of SA-EITC, a concentration representing thelocal concentrations of SA-EITC typically obtained over positive spotson the dilution chips containing dilute amounts of biotin. Both thepolymerization kinetics and the final conversion of monomer to polymerdecrease due to the addition of nanoparticles. The decrease in finalconversion suggests that nanoparticles are capable of terminatingpropagating free radicals during the polymerization process.

An important observation from FIG. 12 is that without the addition ofSA-EITC to monomer, polymerization is only observed at long exposuretimes (greater than 40 minutes, as shown in FIG. 12D). Photoinitiationdue to photoexcited fluorescent nanoparticles at nanomolarconcentrations is thus considerably less efficient than initiation fromSA-EITC. This exposure window where polymerization occurs exclusivelyfrom SA-EITC allows for specificity in the amplification. At lowerexposure times, only surface-initiated polymerization will occur onbiochips due to the presence of SA-EITC from biotin-avidin binding,while unwanted, non-specific initiation from the fluorescent nanosphereswith bulk monomer does not occur until much longer exposures. Thus, thefluorescent monomer system is amenable to on-chip signal amplificationto obtain amplified fluorescent signals.

Finally, amplification using monomer solutions containing nanomolarconcentrations of Nile-Red FluoSpheres from SA-ETIC functionalizeddilution chips were performed. At 20 nm diameter, the nanospheres becomeencapsulated into the PEGDA hydrogel matrix and are not released duringwashing steps, despite lacking covalent attachment into the gel network.Stereomicroscope images of dilution chips showed that beforeamplification, SA-EITC functionalized spots do not produce asignificantly detectable signal. After amplification the last sixcolumns (printed in triplicate) corresponding to higher end biotinconcentrations show an amplified, quantifiable signal. FIG. 13 a detailsthe increase in fluorescent signal from the amplification. The number ofencapsulated nanospheres and the corresponding fluorescent intensityincrease monotonically with film thickness after amplification ondilution chips (nanospheres at 90 and 500 nM concentrations). FIG. 13 bshows measured fluorophore density verses biotin-SA-EITC binding eventsafter polymerization-based amplification. A large gradient influorescent signal ranging from 50 to 8000 Cy3 fluors/μM² is achievedthat corresponds to the number of biotin-avidin binding eventsgenerating the signal. The overall gains in fluorescent signal hererange from 10¹-10². Because the number of SA-EITC binding events thatoccur over a given oligonucleotide concentration can be manipulateddepending on binding conditions, as previously demonstrated, theamplification is reported in terms of binding events/μm² as opposed toDNA markers/μm². This results in variation of amplified fluorescentsignal corresponding linearly to the number of biotin-SA-EITC bindingevents occurring on the surface over an order of magnitude.

Moreover, the gain in fluorescent signal achieved through theamplification allows for characterization of the biotinylated DNAconcentrations using less sophisticated instrumentation. Prior toamplification, SA-EITC functionalized dilution chips were onlydetectable using a microarray scanner employing a PMT detector with lowdetection limits measured at ˜0.28 Cy3 fluors/μm². After theamplification, the dilution chip was characterized using astereomicroscope containing a fluorescent CCD camera, a considerablyless sophisticated, less expensive instrument with much higher detectionlimits measured at ˜570 Cy3 fluors/μm². Currently, the addition of thenanospheres appears to decrease the polymer film thicknesses and thusthe sensitivity of the amplification as opposed to amplification withouttheir inclusion, consistent with the decrease in final conversion ofbulk polymerizations with higher amounts of nanoparticles noted fromFIG. 12.

1. A method for identifying a target comprising the steps of providing aprobe array comprising a plurality of different probes, wherein theprobes are attached to a solid substrate at known locations; the methodcomprising the steps of: a. contacting the target with the probe arrayunder conditions effective to form a target-probe complex; b. removingtarget not complexed with the probe; c. contacting the target-probecomplex with a photoinitiator label under conditions effective to attachthe photoinitiator label to the target-probe complex, the photoinitiatorlabel comprising a photoinitiator capable of being activated by exposureto ultraviolet (UV) light; d. removing photoinitiator label not attachedto the target-probe complex; e. contacting the photoinitiator-labeledtarget-probe complex with a polymer precursor solution; f. exposing thephotoinitiator-labeled target-probe complex and the polymer precursorsolution to UV light, thereby forming a polymer; and g. detecting thepolymer formed, wherein the location of the polymer formed indicates theprobe which forms a target-probe complex with the target, therebyidentifying the target and the oxygen content of the polymer precursorsolution during step f) is limited by contacting the polymer precursorsolution with a purge gas prior to step e), during step e), during stepf), or combinations thereof.
 2. The method of claim 1, wherein thetarget comprises one of biotin or a biotin-binding protein, thephotoinitiator label comprises the other of biotin or a biotin-bindingprotein, and the photoinitiator label is attached to the target-probecomplex by interaction between the biotin and the biotin-bindingprotein.
 3. The method of claim 2, wherein the photoinitiator labelcomprises a plurality of photoinitiators and at least one biotin-bindingprotein attached to backbone of a second polymer.
 4. The method of claim3, wherein the average number of initiators attached to the polymerbackbone is from 100 to
 200. 5. The method of claim 3, wherein thepolymer precursor solution is aqueous and comprises a monomer and acrosslinking agent.
 6. The method of claim 4, wherein in step c) thetarget-probe complex is contacted with an aqueous solution comprisingthe photoinitiator label and the polymer backbone is hydrophilic.
 7. Themethod of claim 6, wherein the polymer backbone is poly (acrylic acidco-acrylamide).
 8. The method of claim 6, wherein the photoinitiator iswater soluble.
 9. The method of claim 6 wherein a blocking agent is usedto minimize nonspecific adsorption of the photoinitiator label on thesubstrate.
 10. The method of claim 9, wherein the target comprisessingle-stranded DNA ((ssDNA) or RNA and the probe comprises ssDNA havinga sequence complementary to at least a portion of the sequence of thetarget.
 11. The method of claim 9, wherein the target comprises one ofan antibody or antigen and the probe comprises the other of an antibodyor antigen.
 12. The method of claim 9, wherein the target comprises afirst protein, the probe comprises a second protein, and the first andsecond protein are capable of molecular recognition.
 13. The method ofclaim 9, wherein the amount of the target can be determined throughcomparison of the polymerization conditions to obtain a selected valueof a detectable characteristic of the polymer to a referencecorrelation.
 14. The method of claim 13, wherein the minimum exposuretime in step f) required to obtain sufficient polymer formation forvisual detection is compared to a reference correlation.
 15. The methodof claim 13, wherein the minimum radiation dose in step f) required toobtain sufficient polymer formation for visual detection is compared toa reference correlation.
 16. The method of claim 9 wherein theamplification factor is at least 1×10⁶.
 17. A method for identifying atarget comprising the steps of providing a probe array comprising aplurality of different probes, wherein the probes are attached to asolid substrate at known locations; the method comprising the steps of:a. contacting the target with the probe array under conditions effectiveto form a target-probe complex; b. removing target not complexed withthe probe; c. contacting the target-probe complex with a photoinitiatorlabel under conditions effective to attach the photoinitiator label tothe target-probe complex, the photoinitiator label comprising aphotoinitiator capable of being activated by exposure to visible light;d. removing photoinitiator label not attached to the target-probecomplex; e. contacting the photoinitiator-labeled target-probe complexwith a polymer precursor solution comprising a polymer precursor and aco-initiator; f. exposing the photoinitiator-labeled target-probecomplex and the polymer precursor solution to visible light, therebyforming a polymer; and g. detecting the polymer formed, wherein thelocation of the polymer formed indicates the probe which forms atarget-probe complex with the target, thereby identifying the target andthe oxygen content of the polymer precursor solution during step f) islimited by contacting the polymer precursor solution with a purge gasprior to step e), during step e), during step f), or combinationsthereof.
 18. The method of claim 17, wherein the target comprises one ofbiotin or a biotin-binding protein, the photoinitiator label comprisesthe other of biotin or a biotin-binding protein, and the photoinitiatorlabel is attached to the target-probe complex by interaction between thebiotin and the biotin-binding protein.
 19. The method of claim 18,wherein the photoinitiator label comprises a plurality ofphotoinitiators attached to a biotin-binding protein.
 20. The method ofclaim 19, wherein the average number of initiators attached to thebiotin-binding protein is from 2 to
 3. 21. The method of claim 20,wherein the initiator is fluorescein or a fluorescein derivative. 22.The method of claim 19, wherein the polymer precursor solution is anaqueous solution and comprises a water soluble difunctional monomer. 23.The method of claim 22 wherein the pH of the polymer precursor solutionis of the polymer precursor solution is greater than 7 and less than orequal to
 9. 24. The method of claim 19 wherein a blocking agent is usedto minimize nonspecific adsorption of the photoinitiator label
 25. Themethod of claim 24, wherein the polymer precursor solution furthercomprises a plurality of detectable particles.
 26. The method of claim25, wherein the detectable particles are nanoparticles encapsulating afluorescent dye.
 27. The method of claim 24, wherein the targetcomprises single-stranded DNA ((ssDNA) or RNA and the probe comprisesssDNA having a sequence complementary to at least a portion of thesequence of the target.
 28. The method of claim 24, wherein the targetcomprises one of an antibody or antigen and the probe comprises theother of an antibody or antigen.
 29. The method of claim 24, wherein thetarget comprises a first protein, the probe comprises a second protein,and the first and second protein are capable of molecular recognition.30. The method of claim 24, wherein the amount of the target can bedetermined through measurement of a detectable characteristic of thepolymer formed.
 31. The method of claim 30, wherein the thickness of thepolymer formed is measured.
 32. The method of claim 25, wherein theamount of the target can be determined through measurement of adetectable characteristic of the detectable particles.
 33. The method ofclaim 32, wherein the detectable particles are fluorescent nanoparticlesand the fluorescence of the nanoparticles is measured.
 34. The method ofclaim 24 wherein the amplification factor is at least 1×10⁶.